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Spindle assembly checkpoint and chromosome stability in Caenorhabditis elegans Tarailo, Maja 2007

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SPINDLE ASSEMBLY CHECKPOINT AND CHROMOSOME STABILITY IN CAENORHABDJTIS ELEGANS  by MAJA TARAILO B.Sc., The University of British Columbia, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA December 2007  © Maja Tarailo, 2007  11  ABSTRACT In many species, proper chromosome segregation is accomplished with the aid of a surveillance mechanism, the spindle assembly checkpoint. To identify the mechanisms involved in this process, the mutants that suppress or enhance the mdf-i (gk2)/MADJ checkpoint lethality were characterized. The suppressors of mdf-i (gk2) fall into two classes. The major class of suppressors compensates for the loss of the checkpoint by delaying mitotic progression. This class includes two known suppressors and anaphase promoting complex/cyclosome (APC/C) components, emb-30/APC4 and fzy-i/CDC2O, and four new such (suppressors of spindle checkpoint defect) genes. One of the new such genes was found to be an APC5-like gene not previously identified as a component of the APC/C in C. elegans. This analysis revealed that APC5 and APCJO genes have paralogs in the C. elegans genome. Furthermore, a class of suppressors was identified that does not delay mitotic progression. In mouse cells, mutations that result in defective apoptosis rescued the lethality associated with deletion of the Mad2 gene. The suppressor mutants were analyzed for the apoptotic response and the such-7(h1985) suppressor with normal anaphase onset was found to abrogate the DNA damage-induced apoptosis. Despite the defective apoptotic response in this suppressor, loss of apoptosis alone could not rescue the mdf1 lethality in C. elegans, indicating that other processes affected by the such-7 mutation, could account for the rescue of mdJi(gk2) lethality. The upstream components required for the genome stability would be recognized as enhancers of the mdf 1 (gk2) lethality. In yeast, 79 genes were found to result in synthetic lethal phenotype with MAD]. Of the 21 non-essential putative C. elegans orthologs assayed, nine enhanced mdJl (gk2) lethality. The enhancers have a specific effect on the SAC, since six of them enhanced the mdf-2(tm2910)/IvIAD2 lethality, three also enhanced the san-i (ok1580) lethality and none enhanced lethality in the kinetochore mutant him-iO(ei5lits)/NUF2. In  111  addition two interactions, hcp-] and bub-3, were identified in C. elegans that are not conserved in yeast. This analysis also showed that HCP-1 and HCP-2, the two CENP-F-related proteins, have a non redundant role, since only the hcp-](RNA1) enhances lethality of the SAC mutants.  iv  TABLE OF CONTENTS ABsTRAcT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  viii  LIST OF FIGURES  ix  LIST OF ABBREvIATIONs  xi  ACKNOWLEDGEMENTS  Xii  DEDICATION  xiii  CO-AuTHoRSHIP STATEMENT  xiv  Chapter 1 1.1  GENERAL INTRODUCTION  1  1.1.1  The Cell Cycle  1  1.1.2  TheMphase  4  1.1.3  The Spindle Assembly Checkpoint (SAC) components  6  1.1.4  The SAC signaling pathway  9  1.1.5  The SAC and Chromosome Instability (CIN)  13  1.1.6  Caenorhabditis elegans: model organism  14  1.1.7  The SAC in C. elegans  15  —  general overview  1.2  THEME AND OBJECTIVES OF THE WORK  19  1.3  BIBLIOGRAPHY  20  Chapter 2: Suppressors of the mdf-1(gk2) Lethal Phenotype Fall into Two Classes 2.1  INTRODUCTION  2.2  MATERIALS  2.2.1  and  29 METHODS  C. elegans strains, alleles and culturing  31 31  V  2.3  2.2.2  Genetic screen for the h2168 suppressor of the mdJ](gk2) lethality  31  2.2.3  Measuring the level of suppression  32  2.2.4  Immunofluorescence  32  2.2.5  Cell division timing of early embryo  33  2.2.6  Phenotypic analysis of the suppressors in wild-type background  33  2.2.7  SYTO12 Staining  34  2.2.8  RNA interference  34  REsuLTs  35  2.3.1  Suppressors rescue the mdJ](gk2) lethality and generate CIN  35  2.3.2  The majority of the suppressors delay mitosis  38  2.3.3  Mitotic delay in Class I suppressors is due to delay in anaphase onset and correlates with securin accumulation  43  2.3.4  The Class II suppressor, h]985, displays an aberrant apoptotic response  46  2.3.5  Neither cep-] nor ced-3 rescue the mdf-](gk2) lethality  49  2.4  DiscussioN  51  2.5  BIBLIoGRAPHY  56  Chapter 3: Characterization and Mapping of the Suppressors 3.1  INTRODUCTION  3.2  MATERIALS  and  58 METHODS  60  3.2.1  C. elegans strains, alleles and culturing  60  3.2.2  Genetic mapping using visible markers  60  3.2.3  Genetic mapping using snip-SNP markers  61  3.2.4  Sequencing  61  3.2.5  DAPlstaining  61  3.2.6  Identification of the h1988ts, h1959ts and h1962ts suppressors  62  vi  3.3  3.2.7  Identification of the such-] (h]960) suppressor  62  3.2.8  RNA interference  62  3.2.9  The UV response and SYTO12 Staining  63  RESULTS  64  3.3.1  Three Class I suppressors are known APC/C components  64  3.3.1.1 The h1988ts suppressor is a new allele offzy-1/CDC2O  64  3.3.1.2 The h]959ts and h1962ts suppressors are new alleles of emb-30/APC4  3.3.2  64  3.3.1.3 The sterility of the emb-30(h]962ts) allele is specific to sperm  66  The such genes identify new mdJ4/MAD] interactors  68  3.3.2.1 such-] (h]960) corresponds to Y66D12A.17, an APC5-like gene  70  3.3.2.2 M163.4/APC5 and FI5HIO.3/APCJO have paralogs in C. elegans genome  72  3.3.2.3 such-2(h]992)  73  3.3.2.4 such-3(h]989)  75  3.3.2.5 such-4(h2]68)  76  3.3.2.6 such-5(h]987)  76  3.3.2.7 such-6(h]958)  76  3.3.2.8 The Class II suppressor, such-7(h]985), abrogates DNA damageinduced apoptosis  77  3.4  DiscussioN  79  3.5  BIBLIoGRAPHY  84  Chapter 4: Enhancers of the mdf-1(gk2) Lethal Phenotype 4.1  INTRoDucTIoN  4.2  MATE1UALS  and METHoDS  87 89  vii  4.3  4.2.1  C. elegans strains, alleles and culturing  89  4.2.2  Measuring the level of enhancement of mdf-i (gk2) lethality  89  4.2.3  Detecting putative orthologs  90  4.2.4  RNA interference assay  90  4.2.5  Measuring viability in md/2, san-i and him-JO mutants  90  4.2.6  Suppression assay  90  REsuLTs  91  Enhanced md/i(gk2) lethality  91  4.3.1.1 Enhanced md/i (gk2) lethality in F 2 generation  91  4.3.1.2 Enhanced md/i (gk2) lethality in F 1 generation  93  4.3.2  Identification of conserved mdf-i/MADJ genetic interactions  93  4.3.3  Enhancement of the other checkpoint mutants  95  4.3.4  Y54G9A.6/BUB3 enhances the lethality of SAC mutants  97  4.3.5  hcp-1/CENP-F enhances the lethality of SAC mutants  99  4.3.6  Ability of md/i suppressors to rescue enhanced md/i lethality  4.3.1  102  4.4  DiscussioN  105  4.5  BIBLIoGRAPHY  112  Chapter 5: Conclusions 5.1  DiscussioN  118  5.2  BIBLIoGRAPHY  126  Appendix A.  Chromosome V G/C-tract analysis in such-4(h2i68) suppressor strain  134  B.  Phenotypic characterization of the such-4(h2i68) suppressor strains  136  C.  BIBLIOGRAPHY  138  viii LIST OF TABLES  Table 1.1  Components of the Spindle Assembly Checkpoint  Table 1.2  The SAC Signaling Pathway  10  Table 2.1  Suppressors rescue the lethal phenotype of mdf-1 (gk2)  36  Table 2.2  The mitotic delay in suppressor mutants is independent of the MDF-1  41  Table 2.3  Suppressor phenotypes in the absence of mdf-1(gk2)  44  Table 2.4  Timing of anaphase onset in the suppressor mutants  46  Table 3.1  Positional cloning of such genes  65  Table 3.2  The emb-30(h1962ts) mutant phenotype  67  Table 3.3  The apc-]O(gk143) mutant phenotype  75  Table 4.1  Genetic interaction between the mdf-]/MAD 1 intero logs and the mdf.2/MAD2  8  and san-1/MAD3 checkpoint components Table 4.2  Suppression of the synthetic lethality of HCP-1- or BUB-3-depleated mdf-](gk2) animals  Table 4.3  98  103  C. elegans genes that enhance the mdf-](gk2) lethality when inactivated by RNA1 109  Table 4.4  Program Predicted Interactions vs. Observed Interactions  111  Table 5.1  SAC pathway genes mutated in human tumours  123  Table A. 1  Analysis of G/C tracts, located on chromosome V, in such-4(h2168) suppressor  Table A.2  strain  135  The such-4(h2]68) strains  136  ix LIST OF FIGURES Figure 1.1  Illustration of the cell cycle  Figure 1.2  Illustration of the five mitotic stages during the first mitotic division of the C.  2  elegans embryo  5  Figure 1.3  Illustration of the SAC pathway  11  Figure 2.1  Mitotic germ cell accumulation in the suppressors  39  Figure 2.2  Timing of cell division at one-cell stage in suppressors  40  Figure 2.3  Timing of cell division at two-cell stage in suppressors  41  Figure 2.4  Apoptotic response in the suppressors  48  Figure 2.5  Neither cep-1 nor ced-3 rescue the mdf-1(gk2) lethality  50  Figure 2.6  Correlating mitotic delay with survivability (%fertile progeny) and genome stability (Him)  52  Figure 3.1  The emb-30(h1962ts) allele has an Emo phenotype at 25°C  69  Figure 3.2  Molecular cloning and DNA sequence analysis of the such-11Y66D12A.17 gene 71  Figure 3.3  Both gfl-3/M163.4 and such-li Y66D12A.1 are required for meiosis  74  Figure 3.4  The such-7 suppressor abrogates DNA damage-induced apoptosis  78  Figure 4.1  dpy-lO(e128) and dpy-5(e51) enhance the mdfl(gk2) lethality  92  Figure 4.2  An integrated H2B::GFP transgene, ruIs32, enhances the mdf-l(gk2) lethality in 1 F  94  Figure 4.3  Enhancers identify conserved mdf-J1MAD 1 genetic interactions  96  Figure 4.4  hcp-l(RNA1) and bub-3(RNAz) enhance the mdf-l(gk2) lethality  100  Figure 4.5  Summary of the genetic interaction data  101  x Figure 5.1  Summary of the synthetic suppressed/enhanced genetic interactions with mdJ1 121  Figure A.1  Viability of three different such-4(h2168) suppressor strains  137  xi  LIST OF ABBREVIATIONS APC/C, anaphase promoting complex or cyclosome CIN, chromosome instability DDR, DNA damage response DAPI, 4’,6-diamidino-2-phenylindole hydrochloride DIC, differential interference contrast Emo, endomitotic oocyte EMS, ethyl methanesulphonate GFP, greenfluorescent protein Him, high incidence of males MCC, mitotic checkpoint complex NEBD, nuclear envelope breakdown NER, nuclear envelope reformation RBH, reciprocal best  BLASTP  hit  RNAi, RNA interference SAC, spindle assembly checkpoint SNP, single nucleotide polymorphism ts, temperature sensitive  xl’  ACKNOWLEDGEMENTS  I would like to start by thanking my supervisor Ann Rose for her patience, guidance and support. Her open mind and curiosity have had a major impact on me. I am also grateful to her for years of generous support and numerous conferences she sent me to, which allowed me to meet many amazing people in this field. I would also like to thank my supervisory committee, Steven Jones, Donald Riddle and Michel Roberge for their guidance and thoughtful comments. I wish to thank in particular David Baillie, Steven Jones and Francis Ouellette for introducing me to the world of bioinformatics; CIHR and Ann Rose for funding my bioinformatics education that was of enormous help during the course of this thesis. I would further like to thank Risa Kitagawa for the encouragement and the selfless help in many aspects of the suppressor project. I am indebted to Riddle and Baillie labs for not only sharing the experimental equipment, but also for providing encouragement and support. I would further like to thank the members of the Rose lab, in particular Sanja Tarailo and Nigel O’Neil who helped shape this thesis and Berdjis Babrami and Shir Hazir for their technical assistance in the study. Most of all, I want to thank the people who kept me motivated through the most difficult times of this thesis. To my parents and my sister, for their unconditional love and endless, complete support that did not allow me to fall even during the hardest times of uncertainty. To my Marko, for coming along at the right time to awake with his kind heart Maja in me again. It is him who makes the best part of my every day. I also am very grateful to my colleagues and best friends, Samuel, Marija, Radka, Maja and Boris for having shared with me joy and pain of graduate school throughout the last years. I want to express my love for my family and friends, who did not allow 10,000km separate them from my life. To them I dedicate this thesis. This work was supported in part by a University Graduate Fellowship.  1 IthnwnJ,4  mx  xiv  CO-AUTORSFIIP STATEMENT  The research described in chapters 2 and 3 was 100% designed, performed and analyzed by me. The publication that resulted from this research also included the analysis of securin levels in the suppressors, which was performed by Dr. Risa Kitagawa, and was not included in this thesis. The manuscript was written by me and edited by co-authors. The research described in chapter 4 was 100% designed, performed and analyzed by me. A part of the research was assisted by the Research Assistant, Sanja Tarailo. The manuscript was written by me and edited by co-authors.  1  Chapter 1 1.1 1.1.1  The Cell Cycle  —  GENERAL INTRODUCTION  general overview “Omnis cellula e cellula”  “Where a cell arises, there must have been a preexisting cell, just as an animal arises only from an animal and a plant only from a plant” (ViRcHow, 1854). The concept of cell theory, also known as cell doctrine, was formally articulated in 1839 by ScHwANN and in 1842 by ScHLEIDEN, and the correct interpretation of cell formation by division was formally enunciated in Rudolph Virchow’s dictum “Omnis cellula e cellula”, which states that perpetuation of life is based on cell division, a complex process that faithfully passes genetic information from one generation to the next. Proper cell division requires a precisely ordered sequence of events in which a cell replicates its DNA and divides into two identical daughter cells. This cycle of cell growth, replication of the genetic material and division is called the cell cycle. The eukaryotic cell cycle is divided into four well-defined stages (Figure 1.1 A). During the DNA synthesis phase (S phase), the cell replicates its nuclear DNA to produce copies for both daughter cells. During the mitotic phase (M phase), the maternal nuclear envelope breaks down, sister chromatids are pulled to opposite poles of the cell, each set of daughter chromosomes is surrounded by a newly formed nuclear envelope, and the cytoplasm splits in two (cytokinesis), producing two identical daughter cells. The somatic cell cycle also contains Gap phases (G phase). The G 1 phase connects the completion of M phase to the beginning of S phase. During G 1 phase, the cell grows and, depending on environmental conditions or developmental signals, either progresses through a commitment point near the end of G 1 and continues through another division or ceases to divide, entering a quiescent or arrested phase known as G . 0  2  A All chromosomes properly attached? DNA damage? Replication Complete?  M itosis  IGoI  Restriction Point  6h 0  T DNA damage? Size? Nutrition?  B Mitosis Embryonic C:ilC:  Endoreduplication Cycle  Figure 1.1: illustration of the cell cycle. (A) Four different cell cycle stages in somatic eukaryotic cells,  M phase (mitosis), G , S phase and (12. M phase consists of nuclear division (mitosis) and cytoplasmic 1 division (cytokinesis). Also included are, G 0 phase (cells that cease division), restriction point (cells that pass this point have committed to S phase) and timing of checkpoint surveillance (1). (B) The cell cycle variations observed during development. The figure was modified from VAN DEN HEUVEL., Cell cycle regulation (September 21, 2005).  3 The G 0 phase may be temporary or permanent. The second gap phase is G 2 phase, which separates S and M phase. During G 2 phase, new proteins are synthesized and the cell continues to grow. During development, eukaryotic organisms use variations of this four-staged somatic cell cycle to fulfill specific developmental requirements (Figure 1.1 B). For instance, early embryogenesis in many organisms is rapid and consists entirely of S and M phases, and lacks gap phases (reviewed in O’FARELL et al. 2004). Endoreduplication, also known as endoreplication, is a process in which S phases are not followed by mitosis (Figure 1.1 B). This process gives rise to cells with extra copies of DNA, permiting amplification of the genome in specialized cells. In mammals, these include myocardial cells; in C. elegans, these include gutcells (reviewed in EDGAR and ORR-WEAvER 2001). Another example of a modified cell cycle is the meiotic cell cycle, a specialized cell cycle that generates haploid gametes and consists of a single S phase that is followed by two consecutive nuclear divisions. The ability of cells to maintain genomic integrity by replicating their DNA with high fidelity and accurately distributing genetic material into daughter cells is crucial for survival and reproductive success of an organism. In order to ensure integrity of the genome, eukaryotic cells employ surveillance mechanisms, known as checkpoints. These include DNA damage, DNA replication and spindle assembly checkpoints, among others (Figure 1.1 A). All known cell cycle checkpoints display a similar organization that includes sensors, mediators, transducers and effectors. Sensor proteins monitor the genome for any defects and help emit a signal that is amplified and propagated by adaptors/mediators and signal transducers to checkpoint effectors. The effectors then regulate a target to delay cell cycle progression in order to provide time for repair of defects. When the damage is beyond repair, cell proliferation may be permanently prevented by programmed cell death, also known as apoptosis, or by senescence. Most, if not all checkpoint pathways are conserved from worm to human.  4 1.1.2  The M phase “wrç” and “psw)cnç”  Waither Flemming, a founder of cytogenetics, investigated chromosome behaviour during cell division and was one of the first scientists to describe in detail numerous events during the process of chromosome segregation. He named this process mitosis (derived from the Greek word iutac (thread), which refers to the thread-like appearance of condensed chromosomes)  (FLEMMING  1882). His observations lead to the extended version of Virchow’s  aphorism, which includes the nucleus: “Omnis nucleus e nucleo “. In principle, during mitosis, one round of DNA replication is followed by a single round of chromosome segregation, thus generating two genetically identical nuclei in the daughter cells. Mitosis can be divided into five distinct stages: prophase, prometaphase, metaphase, anaphase and telophase (Figure 1.2). During the first mitotic division of the C. elegans embryo, maternal and paternal pronuclei migrate towards each other while sister chromatids start to condense during prophase (Figure 1.2). Following nuclear envelope breakdown, prometaphase chromosomes attach to spindle microtubules via kinetochores (proteinaceous structures associated with centromeric DNA), and undergo active movement. As mitosis progresses, more microtubules attach to sister kinetochores leading to a decrease in rapid chromosome movements and ultimate congression to the equator of the spindle, so-called metaphase plate. At metaphase, each sister chromatid has achieved proper bipolar attachment to the microtubules at kinetochores. At anaphase onset, sister chromatids synchronously separate and are pulled away from each other toward the corresponding spindle poles at a high rate. During telophase, the nuclear envelope reforms around the daughter chromosomes and chromosomes begin to decondense. By the end of telophase, the spindle has dispersed, and the cytoplasm has been divided (cytokinesis) creating two identical daughter cells, each with one nucleus (Figure 1.2).  5 Condensing chromosomes Pronuclear meeting Cytoplasm division  Prophase Chromosomes Spindle pole  Prometaphase Telophase and cytokinesis  reformation  Anaphase  Metaphase  Sister chromatids separate  Figure 1.2: fllustration of the five mitotic stages during the first mitotic division of the C elegans embryo. The figure was modified from OEGEMA and HYMAN, Cell division (January 19, 2006).  Unlike mitosis, meiosis generates haploid gametes through a specialized division process that consists of one round of DNA replication followed by two rounds of chromosome segregation with no intervening round of DNA replication. The word “meiosis” comes from the Greek word  jLswa1ç,  meaning “to make smaller,” since it results in a reduction in chromosome  number in the gamete (FARMER and MooRE 1905). The first division, or reductional division meiosis I, involves segregation of the homologous chromosomes, whereas equational division meiosis II resembles mitosis and involves segregation of the sister chromatids. Fusion of the maternal and paternal haploid gametes restores the diploid complement of chromosomes in new,  6 unique progeny (Figure 1.2). The specialized role of the meiotic division is also marked by unique meiotic prophase, during which homologous chromosomes synapse and recombine.  1.1.3  The Spindle Assembly Checkpoint (SAC) Components  During a normal cell cycle, eukaryotic cells replicate and segregate their chromosomes with high fidelity. The precision, a hallmark of the segregation of chromosomes to daughter cells, reflects the remarkable fidelity of the spindle assembly checkpoint that governs this complex error-prone process. The spindle assembly checkpoint (SAC), also known as mitotic spindle checkpoint, metaphase-to-anaphase transition checkpoint, kinetochore attachment checkpoint, chromosome distribution checkpoint or simply the spindle checkpoint, serves as an effective mechanism for self diagnosis. This surveillance system generates a ‘WAIT” signal, which delays the onset of anaphase until all the chromosomes are properly attached to the microtubules at kinetochores. Once all the chromosomes have achieved bipolar attachment, and have aligned at the metaphase plate, segregation proceeds. If this “WAIT” signal is absent, or ignored, the delivery of exactly one copy of a chromosome to each daughter cell is compromised. The resulting daughter cells may have too many or too few chromosomes, a phenomenon termed aneuploidy. In humans, such genomic instability may cause miscarriage, birth defects or promote neoplasia by amplifying oncogenes or by reducing tumor suppressor gene dosage (reviewed in HAssOLD and HuNT 2001; BHARADwAJ and Yu 2004). The first evidence that progression through mitosis is carefully monitored came from studies on drugs that depolymerise microtubules and prolong mitosis in vertebrate cells (BRuEs and CoHEN 1936; ZIEvE et a?. 1980). The molecular components involved in the SAC were identified in two genetic screens in Saccharomyces cerevisiae designed to isolate mutants that fail to arrest in mitosis in the presence of microtubule-depolymerizing drugs, such as nocodazole and benomyl. These checkpoint components include Mad 1, Mad2, Mad3 (mitotic arrest  7 deficient) (LI and MURRAY 1991), and Bub 1, and Bub3 (budding uninhibited by benzimidazole) (Table 1.1) (HoYT et al. 1991). Subsequently, the dual-specific kinase Mpsl (monopolar spindle 1) was also found to play a role in the spindle assembly checkpoint (WEIss and WINEY 1996). Together, these six proteins are still considered the core components of the SAC and are widely conserved throughout the eukaryotic kingdom (Table 1.1) (reviewed in CLEVELAND et al. 2003). The checkpoint is believed to define a signal transduction pathway that responds to either a lack of tension or a lack of microtubule occupancy at kinetochores by transmitting an inhibitory signal that prevents metaphase-to-anaphase transition (WATERs et al. 1998; STERN and MuRRAY  2001; LEw and BURKE 2003; HowELL et a?. 2004). The evidence suggests that  kinetochore plays an essential role in the SAC function. Mutations in centromeric DNA sequences and kinetochore proteins result in a SAC-dependent mitotic delays (SPENcER and HIETER  1992; WANG and BURKE 1995; PANQILINAN and SPENcER 1996; WELLS and MURRAY  1996). Recently, some previously characterized kinetochore proteins such as Ndc8O/Hecl have been implicated in the checkpoint function (Table 1.1) (reviewed in KADuRA and SAzER 2005). In higher eukaryotes, additional proteins also contribute to checkpoint activity, suggesting that in these organisms checkpoint signaling is more complex. These include Rod (rough-deal), ZwlO (zeste-white 10), CENP-E or CENP-F proteins (Table 1.1) (ABRIEu et a?. 2000; ScAER0u et a?. 2001; ENcALADA et a?. 2005). Today, 16 years after the spindle assembly checkpoint was initially recognized, much is still unknown about this complex, non-linear pathway, which is required to delay metaphase-to-anaphase transition until all chromosomes are properly attached to the mitotic spindle. The sensors, the nature of the signals, and the components of transduction pathways that promote the delay are still not fully understood.  8 TABLE 1.1 Components of the Spindle Assembly Checkpoint S. cerevisiae  C. elegans  Vertebrates  Function  Essential in C. elegans?  Madi  MDF-i  MAD1  Binds to and recruits Mad2 to unattached  YES  kinetochores Mad2  MDF-2  MAD2  Part of APC/C inhibitory complex; directly binds  NO  to Cdc2O and Madi Mad3  SAN-i  BUBR1  Part of APCIC inhibitory complex; directly binds  NO  to Cdc2O, Bub3 and CENP-E (in higher eukaryotes); vertebrate BUBRi (Mad3 and Bubi hybrid) Bub 1  BUB- 1  BUB 1  Inhibits Cdc2O by phosphorylation; binds Bub3,  YES  Madi and Cdc2O Bub3  Y54G9A.6  BUB3  Part of APCIC inhibitory complex; binds Bubi  NO  and BUBR1 MpsI  Unknown  MPS 1  Role in recruitment of checkpoint proteins to  NA  kinetochores No clear  Unknown  CENP-E  homologue  Binds and activates BUBRI at unattached  NA  kinetochore  Okpl  HCP-1/2  CENP-F  Unknown  YES’  No clear  CZW-1  ZW1O  Recruits Madl-Mad2 to unattached kinetochores;  YES  homologue No clear  binds ROD and Zwilch F55G1.4  ROD  homologue No clear  YES  binds ZW1O and Zwilch Unknown  Zwilch  homologue Ndc8O  Recruits Madl-Mad2 to unattached kinetochores;  Recruits Madi -Mad2 to unattached kinetochores;  NA  binds ZW1O and ROD NDC-80  HEC-1  Outer kinetochore subcomplex; required for  YES  Madi and Mad2 retention at kinetochore Ipil  AIR-2  Aurora-B  Ensures bipolar orientation; proposed to sense  YES  tension at kinetochores  1  Mif2  HCP-4  CENP-C  Unknown  YES  Unknown  KLP-7  MCAK  Unknown  YES  HCP-1 and HCP-2 are functionally redundant proteins; essential when co-depleted.  9 1.1.4  The SAC Signaling Pathway  The presence of a single unattached or improperly attached kinetochore may generate a diffusible signal that inhibits anaphase onset. Although, the exact nature of the inhibitory signal emitted by the unattached/tension-deprived kinetochores is not known, many of the mechanisms that lead to anaphase onset inhibition by the SAC have been established. One of the main consequences of SAC activation is the inhibition of the anaphase promoting complex/cyclosome (APC/C), a complex molecular machine that is essential for the eukaryotic cell cycle. The APC/C is a large 1 .5-MDa E3 ubiquitin ligase complex, which targets proteins for degradation by ubiquitin-mediated proteolysis (reviewed in PAGE and HIETER 1999). To date, the evolutionarily conserved APC/C has been shown to consist of at least a dozen different subunits (Table 2) (reviewed in PETERs 2006). Although the functions of each individual APC/C subunit are not well understood, it is clear that the most important role of the APC/C is to allow chromosome segregation and anaphase onset by dissolving cohesin, a glue-like molecular structure that keeps sister chromatids together (reviewed in PETERs 2006). The ubiquitin ligase activity of the APC/C is regulated by the WD4O repeat protein Cdc2O/Fizzy (VIsINTIN et al. 1997). Active APC/C catalyses ubiquitination of an anaphase inhibitor called securin Pdsl/Cut2p leading to its destruction through 26S-proteosome-mediated proteolysis. Degradation of securin releases separase Espi/Cutip, a cysteine protease that cleaves the Sccl/Mcdl subunit of the cohesin multiprotein complex in mitotic cells (Table 1.2) (ClosK et al. 1998). Cohesion is established by the cohesin protein complex during DNA replication and persists until chromosome segregation. The proteolytic cleavage of Sccl/Mcdl by separase leads to loss of sister chromatid cohesion and initiation of anaphase (Figure 1.3). Although it is still unclear how defective kinetochore-microtubule attachment is detected and how the inhibition of the APC/C by the checkpoint is precisely achieved, it is known that the SAC acts by binding to Cdc2O and inhibiting the activation of the APC/C  10 TABLE 1.2 The SAC Signaling Pathway Components  S. cerevisiae  C. elegcms  Vertebrates  Function  Essential in C.  elegans? SAC effectors  Apcl  MAT-2/POD-3  APC1  Rpnl/2 homology  YES  Apc2  K06H7.6/APC-2  APC2  Cullin homology; Apci 1  YES  APCIC  and Dod binding Apc4  EMB-30  APC4  WD4O repeats  YES  Cdc27/Apc3  MAT- 1IPOD-5  Cdc27  TPR motif Cdhl binding  YES  Apc5  M163.4/GFI-3  APC5  TPR motif  NO  Cdcl6/Apc6  EMB-27/POD-6  CDC16  TPR motif  YES  Cdc23/Apc8  MAT-3/POD-4  CDC23  TPR motif  YES  Doci/ApclO  F15H1O.3/APC-lO  DOC1/APCIO  Doc domain; substrate  NO  recognition Apcl I  F35G12.9/APC-1 1  APC 11  RTNG-H2 finger; E2  YES  recruitment; E3 activity  SAC effectors  Cdc26  MAT-4  CDC26  Unknown  YES  Unknown  Unknown  APC7  TPR motif  NA  Apc9  Unknown  Unknown  TPR association  NA  Swml  Unknown  APCI3  TPR association  NA  Mnd2  Unknown  Unknown  Amal inhibition  NA  Amal  Unknown  Unknown  WD4O domain  NA  Cdc2O  FZY- 1  CDC2O  WD4O repeats; substrate  YES  CDC2O APC targets  recognition Pdsl  IFY-1  SecurinIPTTG  Inhibits separase  YES  Esp 1  SEP-I  Separase  Cleaves cohesins; allows  YES  anaphase onset Cohesin  Sccl/Rad2 1  5CC-I  RAD21  complex  Sister chromatid cohesion;  YES  cleaved by separase Rec8  REC-8  REC8  Scc3  SCC-3  SCC3/STAG3  YES  Smci  HIM- i/SMC- 1  SMCi  YES  Smc3  SMC-3  SMC3  YES  Unknown  TIM-i  Timeless  YES  Meiosis-specific cohesin  YES  11  A  Unattached!untense kinetochore  Recruitment of the SAC components to kinetochore  Activation of the AC components; making a diffusible inhibitor  1  Inhibiting CDC2O and APCIC BubRl-Bub3-Cdc2O  MCC  OR  Mad2-Cdc2O  OR  B Properly attached kinetochore  1 I I I  SAC silenced  CDC2O activates APCIC  Separase activated; securin destroyed  Cohesins cleaved; sister chromatids separate  Figure 1.3: Illustration of the SAC pathway. (A) In the presence of even a single unattached kinetochore, the SAC components are recruited to the kinetochore and activated to inhibit Cdc2O and APC/C activation, resulting in anaphase onset delay. (B) Once all kinetochores have properly attached to microtubules the SAC is silenced, Cdc2O activates APC/C to ubiquitinate securin. Securin is then destroyed by proteolysis, which leads to activation of separase and cleavage of cohesin. Loss of sister chromatid cohesion leads to anaphase onset.  12 (SuDAKw  et a!. 2001; reviewed in KoPs et al. 2005). Inhibition of the APC/C-Cdc2O by the  checkpoint stabilizes securin and prevents separation of sister chromatids until all of the kinetochores are properly attached to the spindle (Figure 1.3). The kinetochore is believed to play an essential role in the SAC function by acting as a catalytic site for the production of “WAIT”  signal. In vertebrates, immunofluorescence microscopy studies revealed that Mad2  associates with kinetochores that are not bound to microtubules, and disappears when proper attachment is achieved (LI and BENEzRA 1996; CHEN et al. 1996). Later, it was shown that like Mad2 all of the vertebrate Mad and Bub checkpoint proteins bind and act at unattached kinetochores (TAYLoR and McKE0N 1997; CHEN et a!. 1998; TAYLoR et a!. 1998; ABRIEu et a!. 2001). In addition, in mammalian cells it was shown that Cdc2O and several checkpoint proteins, including Mad2 and BubRi, are rapidly bound and released from the unattached kinetochores (HOWELL  et al. 2000; SHAH et a!. 2004), suggesting that unattached kinetochores assemble a  platform that catalyzes the formation of a “diffusible” inhibitor of the APC/C. Although the identity of the in vivo “diffusible” inhibitor(s) remains unknown, Mad2 and BubRl are two candidate checkpoint proteins that have been shown to bind to Cdc2O and inhibit its ability to activate APC/C (FANG et a!. 1998; TANG et a!. 2001; FANG 2002). The compromised localization of Mad2 at kinetochores in the absence of Madi, as well as tight association between the two, suggests that Madi possibly recruits Mad2 to unattached kinetochores (CHEN et a!. 1998; CHEN et a!. 1999). In fact, the crystal structure of the tetrameric Madi -Mad2 core complex revealed that Mad2, upon binding Mad 1, undergoes a dramatic conformational change (SIR0NI et a!. 2002), which is similar in extent to the conformational change required for Mad2 binding and possibly for inhibition of Cdc2O (Luo et a!. 2002). It is clear now that unattached kinetochores have two populations of Mad2; one population is stably associated with Madi, while the other rapidly exchanges with free cytosolic Mad2, suggesting that Madi -Mad2 heterodimer recruits and modifies free Mad2 into an active conformation that can bind to and inactivate Cdc2O (SHAH  13 et al. 2004; HowELL et a?. 2004). In addition, it has been suggested that in a similar manner to Mad2-Madl, BubRi may be recruited to improperly attached kinetochores and activated by Bubi (HOWELL et a?. 2004). Thus, it is possible that Mad2-Cdc2O and/or BubRl-Cdc2O are “diffusible” inhibitor(s) that inhibit the APC/C ligase activity in the presence of spindle damage. However, another checkpoint complex has been isolated and termed mitotic checkpoint complex (MCC). The MCC contains nearly stochiometric amounts of BubRi, Bub3, Mad2 and Cdc2O and has been shown to be about 3000-fold more potent at inhibiting APC/C than Mad2 alone (SuDAKn’4  et a?. 2001). Studies in fission yeast have isolated a similar complex consisting of  Mad3, Bub3, Cdc2O and Mad2 (MILLBAND and HARDwIcK, 2002). The precise roles of the Mad2, BubRi and Bub3 in the anaphase-delaying signal transduction and the role of kinetochores remain unresolved. The molecular role of proteins required for the SAC function, shown in Table 1.1, remains to be elucidated.  1.1.5  The SAC and Chromosome Instability (CIN) Accurate chromosome segregation is essential for cell survival and genome stability.  During meiosis, mis-segregation of chromosomes can result in premature abortion of the fetus or generation of offspring with birth defects such as Down, Patau, Edwards or Klinefelter syndromes (reviewed in HAssOLD and HuNT 2001). In fact, aneuploidy is the most commonly identified chromosome abnormality, occurring in at least 5% of all clinically recognized pregnancies (reviewed in HAss0LD and HuNT 2001). In addition, during mitosis, errors in chromosome segregation can result in CIN, which has been recognized as a hallmark of cancers since Boveri in 1914 (reviewed in BHARADWAJ and Yu 2004; KoPs et a?. 2005). In fact, most solid tumour cells that have been investigated are aneuploid and various cancer cell lines show CIN. It is therefore postulated that CIN contributes to tumorigenesis by amplifying oncogenes or by reducing tumor suppressor gene dosage (reviewed in Kos et a?. 2005).  14 The evidence obtained in recent years implicates weakened SAC in aneuploidy and potentially in tumorigenesis (reviewed in KoPs et al. 2005). In mice, tumour incidence was  significantly increased in animals heterozygous for Mad2 or in animals with severely decreased BubRl levels (MIcHEL et a!. 2001; BAKER et al. 2004). In addition, mice heterozygous for Bub3 and BubRi exhibited elevated susceptibility to colorectal or lung tumours after treatment with carcinogen (BABu et a!. 2003; DAT et al. 2004). The discovery that 2 of 19 colorectal cancer cell lines have mutated Bubi and BubRi genes (CAHILL et al. 1998), simulated search and discovery of mutations in SAC genes (Bubi, BubRi, Madi, Mad2, ZwlO, Zwilch and Rod) in a variety of tumors and tumor cell lines (reviewed in Kos et al. 2005). For instance, Madl/mdf] is mutated in human lymphoid, prostate, lung and breast tumors (NOMOTO et a?. 1999; TsuKAsAKI et a?. 2001). Furthermore, patients suffering with a rare recessive condition called mosaic variegated aneuploidy (MVA), which is characterized by an increase in aneuploidy, growth retardation, microcephaly and childhood cancers have germline mutations in BubRi gene (RANKs et a!. 2004).  1.1.6  Caenorhabditis elegans: Model Organism Caenorhabditis elegans offers several advantages to study the function of spindle  assembly checkpoint. It is a well-established animal model, whose developmental processes are controlled by pathways of gene functions that are analogous to those in mammals (SAIT0 and VAN DEN HEUvEL 2002). C. elegans is effectively an isogenic nematode with both  hermaphroditic and male sexes. Wild-type hermaphrodites have five pairs of autosomes and one pair of sex chromosomes (5A; XX). They produce both sperm and eggs, and are able to selffertilize to produce approximately three hundred progeny. Males have five pairs of autosomes, but one X chromosome (5A; XO) and produce only sperm. When mated to males, hermaphrodites will use the male sperm preferentially, which enables transfer of genetic  15 information between strains. This self-fertilizing hermaphrodite is easy to culture in laboratory  and is transparent at all developmental stages, making it possible to examine cell division in real time. Concerted efforts of the C. elegans community have developed a number of research resources to aid in the determination of the phenotypic consequences of gene malfunction. These include RNA interference (RNAi) technology, RNAi libraries, mutants, promoter-GFP strains, the database of SNP markers, the database of physical interactions, and many other helpful resources  and  information compiled  at the  online  database  known  as WormBase  (http://www.wormbase.org/). Most importantly, the SAC components and the SAC signaling pathway are highly conserved in C. elegans (KITAGAwA and RosE, 1999; OEGEMA et al. 2001; NY5TuL  et al. 2003), making it possible to apply knowledge from other cellular systems to study  the SAC function in a living animal.  1.1.7  The SAC in C. elegans In C. elegans, the SAC components mdfJ/M4DJ, mdJ2/M4D2, san-1/MAD3, bub-]/  BUB] and Y54G9A.6/BUB3 have been identified (Table 1.1) (KITAGAwA and ROSE 1999; NYsTuL  et al. 2003; OEGEMA et al. 2001; TARAILO et al. 2007). MDF-1/Madl, MDF-2/Mad2  and BUB-1/Bubl are required in both yeast and C. elegans to promote mitotic delays in the presence of either chemical or mutational disruptions of the microtubule cytoskeleton (KITAGAwA and ROSE 1999; ENcALADA et al. 2005). During anoxia-induced suspended animation, embryos lacking functional SAN-1/Mad3 or MDF-2/Mad2 fail to arrest the cell cycle  (NYSTUL et al. 2003), suggesting that an oxygen sensing pathway triggers a SAC-mediated mitotic arrest. BUB-1/Bubl also localizes to kinetochores and has an essential role in kinetochore function as well as a potential role in regulating chromatin cohesion (OEGEMA et al.  2001; DEsAI et al. 2003; MONEN et al. 2005). In C. elegans and other metazoans, additional proteins also contribute to checkpoint activity. These include F55G1 .4/ROD, czw-1/ ZW1O, hcp  16 1, 2/ CENP-F and others (Table 1.1) (STARR et al. 1997; BAsT0 et a!. 2000; ScAER0u et al. 2001; DEsAI et a?. 2003; ENcALADA et a?. 2005). Depletion of the checkpoint proteins results in a bypass of the cell cycle arrest in the presence of spindle damage, demonstrating a role in spindle checkpoint function (KITAGAwA and ROSE 1999; ENcALADA et al. 2005). Analysis of an mdJ1 deletion mutant, mdj( 1 (gk2), was the first demonstration of the effects of a defective checkpoint in whole animal development. (KrrAGAwA and ROSE 1999). In contrast to the yeast checkpoint genes, which are essential only in the presence of either chemical or mutational disruptions of the microtubule cyto skeleton (LI and MuRRAY 1991; HoYT et al. 1991; WANG and BURKE  1995), in C. elegans, the checkpoint is essential for long-term survival and fertility  (KITAGAwA  and RosE 1999). In the absence of MDF-1, segregational errors arise and  accumulate resulting in  CIN (KITAGAwA and ROSE 1999). The number of defects observed  increase with increasing numbers of cell divisions, which leads to lethality of the homozygous strain after a couple of generations (KITAGAwA and ROSE 1999).  The C. elegans APC/C components emb-27/CDC]6, emb-30/APC4, mat-]/CDC27, mat-2/APC] and mat-3/CDC23 were isolated in forward genetic screens for temperaturesensitive (ts) embryogenesis defective mutants  (emb, abnormal embryogenesis) (CA55ADA et a!.  1981) and ts maternal effect embryonic lethal (Mel) mutants, which arrest as fertilized one-cell embryos blocked at metaphase of the first meiotic division as metaphase-to-anaphase transition defective  (mat) mutants (GOLDEN et al. 2000; SHAKES et a?. 2003). The other known C. elegans  APC/C subunits, K06H7.6/APC2, Ml 63 .4/APC5, F 1 SR 1 0.3/APC-1 0, F3 5G1 2.9/APC1 1 and B05 11 .9/CDC26, were identified through sequence homologies (Table 1.2) (DAvIS at a?. 2002; DONG at al. 2007). The discovery of the APC/C components in C. elegans has lead to an investigation of the role of these components in the multicellular organism. The majority of the components play an essential role in meiotic and mitotic divisions. Their function during these processes has an important consequence in the development of the worm (reviewed in YEONG  17 2004). In addition, all of the subunits are essential for the survival of C. elegans, with the exception of M163.4/APC5 and FI5H1O.3/APCJO (DAvIs at al. 2002). The authors proposed that the C. elegans apc-5 and ape-JO components do not function as a part of the meiotic APC/C. Furthermore, FuRuTA et al. (2000) showed that the lethality of the mcf] deletion, gk2, can be suppressed by a ts mutation in emb-30(tn377ts)/APC4 (FuRuTA et a!. 2000). Members of the Rose laboratory explored this finding by conducting a screen for suppressors of the mdf-1(gk2) lethal phenotype in search of additional components that function in the metaphase-to-anaphase transition (KITAGAwA et a!. 2002). Ten suppressors were recovered from the screen and one of them, h1983, was shown to encode the homolog of Cdc2O, FZY-l (KITAGAwA et al. 2002). The localization of the FZY- 1 antibody and the phenotypic consequence of the fzy-1 malfunction confirmed the essential function of FZY- 1 during the meiotic and mitotic metaphase-to-anaphase transition (KITAGAwA et a!. 2002). In addition, the orthologue of securin Pdsi, IFY-1 was isolated in a yeast two-hybrid screen with FZY-l as the bait. Inactivation of IFY-i by RNAi revealed an essential role of IFY-1 in chromosome segregation during meiosis I, centrosome duplication and cytokinesis (KITAGAwA et a!. 2002). In C. elegans, the depletion of the separase homolog SEP-i, results in a similar phenotype (SIOMOs et a!. 2001). The identification of IFY- 1 and SEP-i as C. elegans securin and separase pair is further supported by their physical interaction (KITAGAwA et al. 2002). In C. elegans, the cohesin complex components smc-]/SMC], sme-3/SMC3, see 3/SCC3, see-]/SCCJ and rec-8/REC8 have been identified through sequence homologies (Table 1.2) (PAsIERBEK et a!. 2001; CHAN et al. 2003; MIT0 et a!. 2003; WANG eta!. 2003). Depletion of SMC-1, SMC-3, SCC-3 and SCC-1 by RNAi results in premature segregation of chromosomes and aneuploidy (MIT0 et al. 2003). REC-8 appears to function specifically during meiosis (PAsIERBEK et a!. 2001). Recently, TIM-i (homologue of the Drosophi!a clock protein TIMELESS, a circadian rhythm regulator) has been co-immunoprecipitated in a cohesin complex  18 comprising of SMC-1, SMC-3, SCC-3 and SCC-1 (CHAN et al. 2003). Depletion by RNAi and immunofluorescence studies have suggested that TIM-i may be important for targeting non SMC cohesin subunits to the chromosomes; however the precise role of TIM-i is still unknown (CHAN  et al. 2003). The striking conservation of the metaphase-to-anaphase transition pathway  components in C. elegans makes this multicellular model organism an attractive system in which to study the SAC function.  19  1.2  THEME AND OBJECTIVES OF THE WORK  The theme of the research is to increase the overall understanding of the molecular mechanism involved in CIN. Understanding how proper chromosome segregation is achieved is important for two main reasons: defects in genes required for accurate chromosome distribution contribute to tumorigenesis, and one of the most successful strategies of clinical chemotherapy is by targeting the machinery responsible for chromosome segregation. Despite the tremendous progress made in an effort to assemble the molecular picture of the SAC pathway many questions remain unanswered. What is not known is how defective kinetochore-microtubule attachment is sensed, how the inhibitory signal is transmitted, how the inhibition of the APC/C by the MCC is accomplished, or how the checkpoint is silenced. The overall objective of my research is to investigate additional components of the checkpoint cascade by means of identification of novel functional interactors of the MDF- 1 checkpoint component in the nematode C. elegans. In order to achieve this, mutants that rescue the mdJ](gk2) lethality and exhibit high level of CIN were characterized. The hypothesis that mutants that delay anaphase onset could bypass the MDF- 1 checkpoint requirement for survival and fertility was tested. Furthermore, the hypothesis that upstream components required for the genome stability would be recognized as enhancers of the mdf 1 (gk2) lethality was also tested.  20  1.3 ABRIEu,  BIBLIOGRAPHY  A., J. A KAHANA, K. W. WOOD and D. W. 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TARAIL0,  M., R. KITAGAwA and A. M. RosE, 2007a Suppressors of spindle checkpoint defect  (such) mutants identify new mdJ]/M4D1 interactors in Caenorhabditis elegans. Genetics 175: 1665-1679. TAYLOR,  S. S., and F. MCKE0N, 1997 Kinetochore localization of murine Bubi is required for  normal mitotic timing and checkpoint response to spindle damage. Cell 89: 727-73 5.  27 TAYLOR,  S. S., E. HA, and F. McKEON, 1998 The human homologue of Bub3 is required for  kinetochore localization ofBubl and a Mad3/Bub 1-related protein kinase. J. Cell Biol. 142: 1-11. TsuKAsAKI,  K., C. W. MILLER, E. GREENsPuI’J, S. EsHAGHIAN, H. KAwABATA et al., 2001  Mutations in the mitotic check point gene, MAD1L1, in human cancers. Oncogene 20: 3301-3305. VAN DEN HEuVEL,  S., S. Cell-cycle regulation (September 21, 2005), WormBook, ed. The C.  elegans Research Community, WormBook, doi/1 0.1 895/wormbook. 1.28.1, http://www.worrnbook.org. VIRCHOw, VIsIN’rIN,  R., 1854 Zellularpathologie, Bd. VIII. Archiv fUr pathologische Anatomie.  R., S. PRINZ, and A. AMON, 1997 CDC2O and CDH1: a family of substrate-specific  activators of APC-dependent proteolysis. Science 278: 460-463. WANG, F., J. YODER, I. ANT0SHEcHKIN and M. HAN, 2003 Caenorhabditis elegans EVL 14/PDS-5 and SCC-3 are essential for sister chromatid cohesion in meiosis and mitosis. Mol. Cell. Biol. 23: 7698-7707. WANG,  Y., and J. D. BURKE, 1995 Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 15: 6838-6844.  WATERs,  J. C., R. H. CHEN, A. W. MURRAY and E. D. SALMoN, 1998 Localization of Mad2 to  kinetochores depends on microtubule attachment, not tension. J. Cell Biol. 141: 11811191. WEIsS,  E., and M. WINEY, 1996 The Saccharomyces cerevisiae spindle pole body duplication gene MPSI is part of a mitotic checkpoint J. Cell Biol. 132: 111-123.  WELLS,  W. A., and A. W. MuRRAY, 1996 Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast. J. Cell Biol. 133: 75-84.  28 YEONG,  F. M, 2004 Anaphase-Promoting Complex in Caenorhabditis elegans. Mol. Cell Biol.  24: 2215-2225. ZIEvE,  G. W., D. TuRNBuLL, J. M. MuLuNs and J. R. McINTosH, 1980 Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells. Exp. Cell Res. 126: 397-405.  29  Chapter 21: Suppressors of the mdf-1(gk2) Lethal Phenotype Fall into Two Classes 2.1  INTRODUCTION  The spindle assembly checkpoint (SAC) governs the timing of metaphase-to anaphase transition and is essential for genome stability. In contrast to the yeast MAD1 gene, which is essential only in the presence of either chemical or mutational disruptions of the microtubule cytoskeleton (LI and MURRAY 1991; WANG and BuRKE 1995), in C. elegans, mdf 1/MAD1 is essential for long-term survival and fertility (KurAGAwA and RosE 1999). Loss of function of mdf-1/!vL4DJ leads to the accumulation of defects including chromosomal abnormalities, X-chromosome loss or non-disjunction, defective gonad development, and embryonic lethality (KITAGAwA and ROSE 1999). The mutant gk2 contains a deletion within the mdf-1/MADJ gene that results in death of the homozygous strain after three generations. FURUTA  et al. (2000) showed that the lethality of the mdf-1 deletion, gk2, can be  suppressed by a ts mutation in emb-30(tn377ts)/APC4. Analysis of the vulval cell lineages and mitotic index suggested that mitosis was lengthened in emb-30/APC4 mutants and led to the proposal that delayed anaphase onset can bypass the spindle checkpoint requirement for survival and fertility (FURuTA et al. 2000). Our laboratory explored this finding by conducting an ethyl methanesulfonate (EMS) mutagenesis screen for suppressors of the mdJ1 (gk2) lethal phenotype in search of additional components that function in the metaphase-to-anaphase transition pathway (KITAGAWA et a?. 2002). A collection of ten mutants capable of rescuing the mdf-1 (gk2) loss of function phenotype was isolated. One of the suppressors, h1983, was shown to encode the  ‘A version of this chapter has been published: TARAIL0,  M., R. KITAGAWA and A. M. RosE, 2007 Suppressors of spindle checkpoint defect  (such) mutants identify new mdJ]/MADJ interactors in Caenorhabditis elegans. Genetics 175: 1665 1679. —  30 homolog of Cdc2O, FZY-1  (KITAGAwA  et al. 2002). The fzy-1(h1983) hemizygotes had a  prolonged metaphase phenotype supporting the hypothesis that delayed anaphase can rescue the mdf 1 (gk2) lethality. In this study, the mechanism of rescue of the mdf] (gk2) lethality, including timing of cell cycle progression, has been investigated in all of the mutants that bypass the MDF 1 checkpoint requirement.  31  2.2 2.2.1  MATERIALS AND METHODS  C. elegans strains, alleles and culturing The Bristol strain N2 was used as the standard wild-type strain (BRENNER 1974). The  marker mutations and balancer chromosomes used are listed in the order of chromosomes. LGI: dpy-5(e61), dpy-5(s]300); LGII: dpy-]O(e]28), unc-4(e120); LGIII: dpy-i 7(e]64), dpy 18(e364), unc-32(e289), LGIV: dpy-13(e]84) and LGV: unc-46(e]77), mdfi(gk2) and nTi(IV;V). The strains used include: VC13 dog-i (gki 0), KR3627 unc-46(e177) mdf-1(gk2) +/+ + nTi [let-X] and DG627 emb-30(tn377ts). The AZ212: unc-]19(ed3) ruIs32[unc-119(+) pie i::GFP::H2B] III strain was used to visualize the chromosomes in the suppressor mutants. The strains were obtained from the Caenorhabditis Genetics Center. Animals were maintained using standard procedures (BRENNER 1974). 2.2.2  Genetic screen for the h2168 suppressor of the mdf-1(gk2) lethality Suppressors of the mdf-](gk2) lethality were isolated in two separate genetic screens.  All the suppressors except for such-4(h21 68) were isolated in the EMS screen as described by KITAGAwA  et al. (2002). The such-4(h2i68) suppressor was isolated using the dog-i (gki 0)  mutator strain (CHEuNG  et al. 2002). A strain of genotype unc-46(ei 77) mdJi (gk2) +/+ +  nTi[let-X]; dog-i(gk]0)/dog-i(gkio) was constructed. The F 1 unc-46 mdf-i homozygotes (n=40) were picked and plated individually. After four weeks a single plate containing fertile worms was isolated as a suppressor candidate. The suppressor was out-crossed from the dog i(gkiO) background to avoid further accumulation of mutations and the strain of genotype unc 46(ei 77) mdf-i (gk2) such-4(h2i 68)/ unc-46(ei 77) mdf1 (gk2) such-4(h21 68) was generated. These worms were crossed to N2 males and re-segregated several times to eliminate secondary mutations generated in the dog-i (gki 0) background.  32 2.2.3  Measuring the level of suppression For analysis of the KR3627 strain (unc-46(e177) mdf-1(gk2) +/+ + nTl [let-X]), five  L4 wild-type looking worms were individually plated at 20°C and 25°C. The worms were transferred to fresh plates every 12 hours and the plates were scored. The eggs that did not hatch in 24 hours were scored as embryonic arrest. The eggs that hatched but did not reach adult stage were scored as larval arrest. The progeny that developed to adult stage were scored for the ratio of wild-type to unc-46 mdf1 worms. The observed over expected percentage at 20°C was 97% and at 25°C was 107%, which indicated that F 1 mdf-1(gk2) homozygotes segregated from heterozygous parents developed normally at both temperatures. Similarly, the F 2 generation was examined by plating 14 unc-46 mdf-1 worms at L4 stage and analyzing the progeny. The percent fertility was determined by individually plating all progeny that developed to adult stage. The suppressors were characterized in the same manner by plating a minimum of 14 suppressed unc 46 mdf1 worms at L4 stage and scoring for embryonic arrest, larval arrest, adult progeny and adult fertile progeny. 2.2.4  Immunofluorescence For antibody staining, http://www. genetics.wustLedu/tslab/protocols.html publicly  available protocol was used. Dissected gonads were fixed with 3% formaldehyde, 0.1M K 4 H 2 PO (pH 7.2), for lh and post-fixed with cold 100% methanol for 5 mm. After re-hydration with lx PBS-T (1X PBS containing 0.1% Tween-20) the samples were incubated at 4°C overnight with anti-phosphohistone H3 antibody (Upstate Biotechnology) at 1:200 dilution to visualize mitotic chromosomes. The samples were then incubated with Oregon-green-conjugated-anti-rabbit IgG antibody (Molecular Probes) at dilution  1:1000.  DAPI (4’,6-diamidino-2-phenylindole  hydrochloride) (1 ig/ml) was added to the secondary antibody solution. The stained gonads were viewed with the Zeiss Axioscope fluorescent microscope with 40X objective. A Retiga 2000R camera (Qimaging) and Openlab 4.0.2 software (Improvision) were used to acquire images.  33 2.2.5  Cell division timing of early embryo One day old adult gravid hermaphrodites were dissected and embryos were mounted in  M9 buffer (BRENNER 1974) between a coverslip and a 3% agarose pad. Mineral oil was used to reduce evaporation at the edge of the coverslip. Embryos were observed using a Zeiss Axioscope at 40X magnification. All images were taken with a Retiga 2000R (Qimaging) digital camera and Openlab 4.0.2 software (Improvision). Time points measured were from the nuclear envelope breakdown (NEBD) to nuclear envelope reformation (NER) as determined by the analysis of DIC images, as described previously (ENCALADA et al. 2005). To measure the time points from NEBD to anaphase and from anaphase to NER, embryos from hermaphrodites carrying an integrated H2B::GFP transgene were prepared and visualized using the same procedure. 2.2.6  Phenotypic analysis of the suppressors in wild-type background The suppressor mutations were linked to Dpy (Dumpy) markers and crossed to N2  males to separate the such mutations from the mdf.1(gk2) background. In all of the suppressors that were successfully separated from the mdf-](gk2) background the absence of the gk2 deletion was confirmed by PCR amplification. Since Dpy markers often interfered with the suppressor phenotypes, one more round of crosses to N2 males was then performed to isolate the such mutations from the Dpy backgrounds. All of the suppressors isolated in wild-type background were analyzed at 20°C and 25°C by plating 10 L4 hermaphrodites and scoring their progeny for embryonic and larval arrest, incidence of males, somatic defects, sterility or developmental delay. The majority of suppressors displayed a more severe phenotype at 25°C; however, only the suppressors that could be maintained at 20°C and were lethal at 25°C were named conditional ts alleles.  34 2.2.7  SYTO12 staining One day old adult hermaphrodites were incubated for 3h in the dark in 50 j.tl of 33 jiM  SYTO12 solution (Molecular Probes, Eugene, OR) in M9 buffer. Animals were then destained by allowing them to feed on NGM 0P50 seeded plates. After lh, animals were mounted in 5mM levamisole in M9 buffer on agarose pads and investigated using a Zeiss Axioscope fluorescent microscope at 40X magnification, as described previously (GuMIENNY et at. 1999). 2.2.8  RNA interference The cep-] bacterial RNAi feeding strain was grown in LB broth containing 5OjigImL  of ampicillin and 5Ojig/mL of tetracycline at 37°C overnight. The bacteria were then streaked on Nematode Growth Media (NGM) plates containing 25jig/mL ampicillin, 12.5 jig/mL tetracycline and 1mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The next day, wild-type animals from the KR3627 strain in L4 stage were plated and fed dsRNA-expressing bacteria. Their progeny of Unc-46 phenotype were collected and analyzed using SYTO12.  35  2.3  2.3.1  RESULTS  Suppressors rescue the mdf-1(gk2) lethality and generate CIN The mdf-1/MADJ gene is essential for the long-term survival and fertility of C. elegans  (KITAGAwA and RosE 1999). mdf-1 homozygotes segregated from the sma-8(n716) +/+ mdf ](gk2)(V) heterozygous parents had no obvious phenotype at 20°C, most likely due to the presence of maternally supplied MDF-1 protein. However, only 21% of F 2 mdf-1(gk2) homozygotes grew to adulthood (KITAGAwA and ROSE 1999). In accordance with this data it was observed that unc-46 mdf-] homozygotes segregated from unc-46(e177) mdf-J(gk2) +/+ + nT][let-XJ heterozygote parents appeared to be normal and grew to adulthood at 20°C. Similarly, only 21% of F 2 unc-46 mdJl homozygotes developed into adults and the majority of the F 3 homozygotes arrested at different developmental stages (Table 2.1). It was also observed that higher temperature intensified the effect; at 25°C, 97% of the F 2 homozygotes arrested before hatching and the remaining 3% arrested at early larval stages. Thus, the unc-46 mdf] homozygotes cannot be maintained beyond the first generation at 25°C, while at 20°C the strain cannot be maintained beyond the third generation (Table 2.1) (TARAIL0 et al. 2007). In a previous (ethyl methanesuiphonate) EMS mutagenesis screen members of the Rose laboratory isolated 10 suppressors of the mdJl (gk2) lethal phenotype (KITAGAwA et a?. 2002). The h2]68 suppressor allele was isolated in the dog-i (gki 0) mutator strain (TARAIL0 et a?. 2007; APPENDIx A). When dog-] is mutated or disrupted by RNA interference (RNAi), deletions frequently initiate at dC/dG-tracts that are spaced throughout the genome and typically extend a few hundred base pairs upstream of the G-tract (CHEuNG et a?. 2002). One plate containing a suppressor mutation was isolated from 80 haploid genomes screened (Table 2.1). To examine the viability of the suppressor mutations in the absence of the MDF- 1 checkpoint, all of the suppressor strains were analyzed for embryonic arrest, larval arrest, adult and fertile progeny  36 TABLE 2.1 Suppressors rescue the lethal phenotype of mdf-1(gk2) Adult  Embryonic  Larval  arrest  arrest  (%)  (%)  (%)  (%)  (%)  2 unc-46(e177) mdf-i(gk2) (n4,133) F  25.2  53.4  21.4  2.5  5.1  2 unc-46(e177) mdf4(gk2) (n=924) 25°C F  97.3  2.7  0.0  0.0.  0.0.  unc-46(e177) mdf-i(gk2); h1959ts (n=829)  14.6  16.4  69.0  57.1  1.5  unc-46(e177) mdf-i(gk2); emb-30(tn377ts)(n=1,740) 16°C  11.4  16.5  72.1  50.4  0.7  unc-46(e177) mdf-i(gk2); h1962ts (n=455)  19.4  14.5  66.1  48.1  3.1  unc-46(e177) mdf-i(gk2); h1960 (n=353)  23.8  15.3  60.9  41.6  0.8  unc-46(e177) mdf-1(gk2); h1958 (n=585)  24.9  22.2  52.9  40.6  1.8  unc-46(e177) mdf-i(gk2); hi988ts (n=470)  28.8  28.9  42.3  33.9  2.5  unc-46(e177) mdf-l(gk2) hi 992 (n=1,135)  45.3  15.0  39.7  30.7  1.8  unc-46(e177) mdf-1(gk2); hi 987 (n=214)  42.0  17.8  40.2  29.0  6.2  unc-46(e177) mdJI(gk2);fzy-i(hi983)(n=1,437)  71.2  6.9  21.9  18.4  1.9  unc-46(ei77)mdf-i(gk2)hi985Qi1,102)  51.8  19.4  28.8  16.7  4.4  unc-46(e177) mdf-i(gk2); hi 989 (n=989)  70.1  9.3  20.6  12.6  3.9  unc-46(e177) mdf-i(gk2) h2168 (n=372)  58.9  24.9  16.2  9.6  9.1  Genotypes  Fertile  Male  adult  Shown here are the phenotypes of the eleven mutants recovered in the screens for suppressors of the mdf 1(gk2) lethal phenotype. Also included are the two previously known suppressors emb-30(tn377ts) and fzy-1(h1983). The extent of suppression was determined at 20°C after at least three backcrosses, unless otherwise indicated.  37 (Table 2.1). The strongest suppressor allele, h]959ts, decreased both the percentage of embryonic arrest and larval arrest when compared to the F 2 unc-46 mdf-1 homozygotes, increasing the percentage of adult progeny (Table 2.1). On the other hand, the weakest suppressor alleles, h1989 and h2]68, resulted in an increase in embryonic arrest and decrease in larval arrest and yielded the same percentage of adult progeny as the 2 F unc-46 mclf 1 homozygotes (Table 2.1). However, all of the suppressor alleles increased the percentage of fertile progeny from 4- to 23-fold when compared to the F 2 unc-46 mdf-1 homozygotes alone (Table 2.1). This analysis suggests that while none of the suppressors rescues the mdJl (gk2) associated phenotypes completely, all of the suppressors allow the strains to be propagated in the absence of the essential SAC component by increasing viability (Table 2.1) (TARAIL0 et al. 2007). The fidelity of chromosome segregation in the suppressor strains was tested by scoring the frequency of spontaneous males. C. elegans populations consist largely of self-fertilizing hermaphrodites (5A; XX). During meiosis I, non-disjunction of the X chromosome in the hermaphrodite germ line generates gametes with no X chromosome, leading to XO male progeny (5A; XO). Males arise spontaneously at a rate of 0. 1% at 20°C (HoDGKIN et al. 1979; RosE and BAILLIE  1979). The members of Rose laboratory have shown previously that F 2 mdJ4  homozygotes have a high incidence of males (Him) phenotype (KITAGAwA and RosE 1999). In the F 2 unc-46 mdf 1 homozygotes a 50-fold increase in the frequency of males was observed when compared to wild-type animals (Table 2.1). The majority of the suppressor mutations substantially decreased X-chromosome mis-segregation in the absence of the MDF- 1 checkpoint. However, none of the suppressors rescued the mdf-1 (gk2)-associated Him phenotype completely (Table 2.1) (TARAILO et al. 2007).  38 2.3.2  The majority of the suppressors delay mitosis One explanation for the ability of the suppressors to bypass the MDF-1 checkpoint  requirement is that they delay anaphase onset.  FuRuTA  et al. (2000) showed that in emb  30(tn3 77ts) gonad arms, there is a significantly increased number of mitotic cells at permissive temperature (16°C), suggesting a mitotic delay in the germ cells. The second identified suppressor,fzy-1(h1983), when analyzed as a hemizygote had a higher mitotic index, suggesting prolonged mitosis infzy-](h]983) hemizygotes  (KITAGAwA  et a?. 2002). To determine whether  all of the suppressors delay mitotic progression, the chromosomes in nuclei undergoing mitosis in the distal mitotic zone of gravid hermaphrodite gonads were visualized by staining with an antibody against phosphorylated histone H3 (Figure 2.1 B). In accordance with the previous data, the unc-46(el 77) mdf] (gk2); emb-30(tn3 77ts) and unc-46(el 77) mdf-1 (gk2); fzy-] (h1983) homozygotes had a significantly increased number of nuclei stained with this antibody compared to wild-type or unc-46(e] 77) mdf] (gk2) animals (Figure 2.1 A). Importantly, seven additional suppressors displayed a variable but significant increase in the number of mitotic cells in the distal arm, suggesting that germ cell mitosis is slowed in these animals (Figure 2.1 A). However, three suppressors displayed a normal number of stained cells suggesting normal cell cycle progression. These data suggest that germ cell mitosis is slowed in the majority of the suppressors. The timing of mitosis in the early embryo was examined next. Recent work from ENcALADA  et a?. (2005) has shown that chemical or mutational disruptions of the mitotic spindle  activate the spindle checkpoint to delay progression through mitosis in rapidly dividing C. elegans embryonic cells. These results suggested that mdf4 is required for the cell cycle delay in early embryonic cells in the presence of spindle damage, which prompted us to analyze the timing of mitotic division in unc-46(e] 77) mdfl(gk2); emb-30(tn377ts) and unc-46(el 77) mdf ](gk2);fzy-1(h]983) early embryos. The mitotic progression from nuclear envelope break down  39  B  WT  t  0  p-histone  U  .-  I: , “  _  _ —  •  ,ø.-  _  _  —  A’  _  ,  — _  ‘‘  I  —  jtP  if’  %i  6’  A’  — ,  A’  si  h1960; mdf-1  DAPI  p-hiStOfle  h1992 mdt-1  fl1992 mdI-1  —‘_ ,_  j  e  A’ )  h1960; mdf•1  1’  DAP)  —  p-histone  Figure 2.1: Mitotic germ cell accumulation in the suppressors  (A) Quantitation of mitotic nuclei in the distal zone of gravid hermaphrodite gonads. Bars represent the mean number of mitotic nuclei per gonad arm with SEM error bars; n = number of gonad arms scored for each suppressor. (B) The representative images of DAPI and anti-phosphohistone H3 stained wild-type, unc-46(e177) mdJl(gk2); h1960 and unc-46(e177) mdf-i(gk2) hi 992 gonads. All the measurements were performed at 20°C. Scale bar represents l0m.  (NEBD) to nuclear envelope reformation (NER) was measured using time-lapse differential interference contrast (DIC) imaging of early embryonic cells as described by ENcAiIA et a!. (2005). In the unc-46(e177) mdf-1(gk2); emb-30(tn377ts) embryos the duration from NEBD to  NER in P0, AB and P1 cells was increased approximately two-fold when compared to wild-type embryos at 16°C (Figures 2.2 and 2.3 and Table 2.2). In addition, the average time required to progress through mitosis was increased 1.8—fold in the unc-46(e177) mdJ1(gk2), fzy-1(h 1983) homozygous one- and two-cell stage embryos when compared to wild-type embryos developing at 20°C (Figures 2.2 and 2.3 and Table 2.2). The mitotic delay observed in the early embryonic  40 A  B  D  Significant difference  No significant difference  WI  0  100  200  300  ‘H 400  500  500  700  Duration from NEBD to NER (seconds)  Figure 2.2: Timing of cell division at one-cell stage in suppressors  (A-C) Images of the wild-type, unc-46(e177) mdJl(gk2) h1985 and unc-46(e177) mdfil(gk2); fzy 1(h1983) one-cell embryos using lime-lapse microscopy. The T  =  0 miii time point shows cells during  NEBD. The embryos were imaged every 2 miii from this point until the formation of the two-cell embryo. Pictures were taken every 2 mm (Scale bar: 2Opm). (D) Summary of the mitotic timing data from live cell imaging. Bars represent the duration of mitotic division from NEBD to NER in one-cell embryos. Mean durations are plotted in seconds with SEM error bars (n=l0 measurements for each strain). The braces depict the controls, the suppressors that do not cause a significant delay in mitotic progression and the suppressors that significantly delay the mitotic liming as shown by p values of the student’s t-test statistic (Table 4) All the measurements were performed at 20°C, except for emb-30(tn377ts) and wild-type control strains observed at 16°C.  41 A  600  D  AB 0 0  P1  500  400  z 0  300  200  100  Figure 2.3: Timing of cell division at two-cell stage in suppressors (A-C) Images of the wild-type, unc-46(e177) mdf-i(gk2) hi985 and unc-46(e177) mdJl(gk2); fzy i(h1983) two-cell embryos from Figure 2. The T =0 mm time point shows cells during NEBD in the AB cell. The embryos were imaged every 2 mm from this point until the formation of the four-cell embryo.  (Scale bar: 20j.tm). (D) Summary of the niitotic timing data from live cell imaging. Bars represent the duration of mitotic division from NEBD to NER in two cell embryos. Mean durations are plotted in seconds with SEM error bars (n=’lO measurements for each strain). The data for h1987, h1958 and hi 985 as well as the previously identified suppressors emb-30(1n3 77ts) and fzy-i (h1983) are included. All the  measurements were performed at 20°C, except for emb-30(tn3 771s) and wild-type control strains observed at 16°C.  42 TABLE 2.2 The mitotic delay in suppressor mutants is independent of the MDF-1 Embryo (genotypes)  NEBD-NER(s)  ±  SE  (n)  t-test Wild  t-test F 2 mdf  type  1(gk2)  Wild-type (N2)  287.3  ±  7.0 (10)  1.0  0.067  F2 unc-46(e177) mdJI(gk2)  304.1  ±  7.8 (10)  0.067  1.0  Wild-type (N2) 16°C  335.3  ±  7.8 (10)  0.00 15*  0.047  unc-46(e177) mdJl(gk2); emb-30(tn377ts) 16°C  651.3  ±  14.3 (10)  * 8 I.0X10  * 8 5.0X10  emb-30(tn377ts) 16°C  627.7  ±  22.2 (10)  * 7 2.4X10  * 7 3.1X1W  unc-46(e177) mdJI(gk2); h1960  628.9  +  16.0 (10)  * 9 7.4X10  * 8 2.3X10  h1960  596.6* 10.7(7)  5.3X10?*  * 7 1.6X10  unc-46(e177) mdJl(gk2);fzy-I(h1983)  543.3  16.3 (10)  * 7 3.57X10  * 7 9.0X10  fzy-1(h1983)  515.2  3.1 (10)  3.l9X10°*  5.59X10°*  unc-46(e177) mdf.I(gk2); h1959ts  484.4  24.8 (10)  * 5 3.8X10  I 4 1.1X10 * 3 4.3X10  ± ± ±  h1959ts  427.3± 13.8 (5)  * 3 5.0X10  unc-46(e177) mdJ4(gk2) h1992  421.3  6.7 (10)  * 6 1.1X10  * 6 2.OXltY  mdJ1(gk2) h1992  397.0± 11.5 (5)  * 4 5.1X10  * 5 s.3x10  unc-46(e177) mdJI(gk2); h1962ts  413.8  13.2 (10)  * 5 1.5X10  1.0X10’  h1962ts  398.6± 18.7 (10)  * 4 2.5X10  * 3 2.1X10  unc-46(e177) mdf-I(gk2); h1989  391.2 ± 9.4 (10)  7.0X1O*  * 5 7.2X10  unc-46(e177) mdf-J(gk2); h1988ts  378.0± 13.0 (10)  * 4 1.5X10  * 3 2.3X10  h1988ts  380.6± 10.2 (5)  * 4 2.5X10  * 4 4.0X10  unc-46(e177) mdf-I(gk2) h2168  357.7± 14.7 (10)  0.0019*  0.021*  unc-46(e177) mdJI(gk2); h1987  324.7± 10.2 (10)  0.018*  0.071  8.5 (5)  0.70  0.67  12.7 (10)  0.19  0.77  h1987  ±  ±  291.8  ±  unc-46(e177) mdJI(gk2); h1958  309.6  h1958  296.6  ±  10.0 (5)  0.20  0.25  unc-46(e177) mdf-J(gk2) h1985  293.1  ±  5.3 (10)  0.29  0.22  ±  Table 2.2 shows statistical analysis of the data sets where NEBD to NER was measured in P0 cells of the suppressors in both wild-type and unc-46(el 77) mdf-1(gk2) backgrounds.  43 cells in these two suppressors correlates with an increase in mitotic nuclei in the germline (Figures 2.1 and 2.2). The NEBD to NER interval duration in all of the suppressors in the unc 46(e] 77) mcf](gk2) background was measured next (Figure 2.2D). The seven suppressors that displayed a significantly elevated number of mitotic nuclei in the germline also progress through mitosis in one-cell embryo with delays (Figure 2.2D). In some mutant embryos, such as h2168, the mean mitotic time was slightly increased to 1.2-fold, while in others, such as h1960, a more dramatic delay of a 2.1-fold increase for the same interval was observed (Figure 2.2D). Interestingly, the h]987, h]958 and h]985 suppressors have normal timing of mitotic progression in early embryonic cells as well as a normal number of mitotic nuclei in the germline (Figures 2.1—3; and Table 2.2). These data show that the mitotic timing in the early embryo correlates with the mitotic timing in the germline (Figures 2.1 and 2.2). In order to test the mitotic delay in the suppressor mutants alone, all the recessive suppressors (with the exception of two mapping to LGV) were isolated from the unc-46(el 77) mdf-1(gk2) background (Table 2.3). All of these suppressors are viable at 20°C and display a range of different phenotypes (Table 2.3). As expected, the mitotic delay in these mutants is independent of the checkpoint (Table 2.2). Thus, this analysis suggests that the suppressors of the mdJl (gk2) lethal phenotype fall into two classes: Class I suppressors delay mitotic division, and Class II suppressors progress through mitosis normally. The suppressors with slowed mitotic cell cycles display mitotic delays in both embryonic and germ cells that may compensate for the absence of the essential checkpoint function.  2.3.3  Mitotic delay in Class I suppressors is due to delay in anaphase onset and correlates  with securin accumulation To determine whether the mitotic delays observed in the suppressor mutants are due to a delay in anaphase onset, suppressor mutant strains that carry ruIs32, an integrated H2B::GFP  44 TABLE 2.3 Suppressor phenotypes in the absence of mdf-1(gk2) Genotypes  Class  h1959ts  RECESSIVE  Phenotypes Viable at 20°C, some embryonic arrest and mild Him; Lethal at 25°C, 100% embryonic arrest  h1962ts  RECESSIVE  Viable at 2 0°C, majority of brood consists of unfertilized oocytes and mild Him; Sterile at 25°C, 100% unfertilized oocytes in F 2  h1960  RECESSIVE  Viable at 20°C, no obvious phenotype; Viable at 25°C, embryonic arrest and Him  h1958  RECESSIVE  Viable at 20°C, developmental delay; Viable at 25°C, some developmental arrest and developmental delay  h1988ts  RECESSIVE  Viable at 20°C, small brood size, embryonic and larval arrest and Him; Sterile at 25°C, 100% sterile  h1992  RECESSIVE  Viable at 20°C, no obvious phenotype; Viable at 25°C, embryonic arrest and Him  h1987  RECESSIVE  Viable at 20°C, no obvious phenotype; Viable at 25°C, developmental arrest  h1985  RECESSIVE  Difficulty isolating due to linkage to unc-46 and mdJI  h1989  DOMINANT  Difficulty isolating due to phenotype  h2168  RECESSIVE  Difficulty isolating due to linkage to unc-46 and mdj4  All of the recessive suppressors (except for h2168 and h1985 located on LGV) were isolated from the  unc-46(e177) mdf-1(gk2O) background and analyzed for phenotypes. The temperature sensitivity was examined by shifting L4 hermaphrodites to 25°C and scoring the progeny.  transgene were constructed (PRAITIs et a?. 2001). In these strains, the mitotic progression from NEBD to anaphase onset and from anaphase onset to NER was analyzed in early embryonic cells as described by ENCALADA et a?. (2005). Seven suppressor strains were successfully constructed; however, the remaining suppressors could not be constructed with ruIs32 either because the  45 H2B::GFP insert is closely linked to the suppressor mutation or because the H2B::GFP transgene is inviable with these suppressor mutants (Table 2.4). The mitotic delay observed in Class I suppressors is solely due to the delay in anaphase onset. For instance, the 1.6-fold delay in the NEBD to NER interval in h]988ts suppressor mutant is due to the two-fold lengthening of the interval from NEBD to anaphase onset (Table 2.4). In contrast, no significant difference was observed in the progression from anaphase onset to NER in any of the suppressors when compared to wild-type and mdf](gk2) embryos with the H2B::GFP insert (Table 2.4). Our data also show that the class of suppressors that display normal mitotic timing in the germline and embryonic cells progress through anaphase with no delay. The mechanism of anaphase onset timing in the suppressors was tested in collaboration with Dr. Risa Kitagawa. Previously, using yeast two-hybrid analysis, KITAGAwA et al. (2002) showed that one of the suppressors isolated in the EMS screen, fzy-1(h1983), loses the binding affinity of FZY- 1 /Cdc2O for securin, IFY- 1 /Pds 1. It was reasoned that the suppressor mutations with delayed mitosis may be defective in securin IFY-1 destruction, which would delay the activation of separase and hence anaphase onset. To test this, the IFY- 1 levels were analyzed by Dr. Kitagawa in the cell extracts prepared from the suppressor mutants  (TARAILO  et a!. 2007).  Western blotting showed IFY- 1 accumulation in the Class I suppressors that delay anaphase onset. On the other hand, the class II suppressors that progress through mitosis normally do not accumulate securin. Furthermore, quantitation of the data revealed that the IFY- 1 protein level correlates with the timing of anaphase onset  (TARAIL0  et a!. 2007). These data suggest that the  Class I suppressors might bypass the checkpoint requirement by reducing the APC/C activity, which results in securin accumulation and delayed anaphase onset. The Class II suppressors most likely rescue the checkpoint lethality by a different mechanism.  46 TABLE 2.4 Timing of anaphase onset in the suppressor mutants Embryo  NEBD-anaphase (s) ± SE (n)  Wild type  154.5  F2 unc-46(e177) mdf-](gk2)  177.2  anaphase  —  NER  (s) ± SE (n)  NEBD-NER (s) SE (n)  ±  4.3 (6)  143.2  14.2 (5)  127.6± 6.1 (5)  304.8± 12.9(6)  unc-46(e177) mdf-I(gk2); h1960  464.2± 13.6(5)*  124.2 ± 2.6 (5)  588.4  unc-46(e177) mdf-1(gk2); h1983  352.2  5.9 (5)*  158.2± 3.3 (5)  510.4± 5.3 (5)*  11.2 (5)*  144.6± 11.4(5)  397.4 ± 7.5 (5)*  344.0± 14.1 (5)*  137.8± 8.1 (5)  481.8  124.6  302.6 ± 8.5 (5)  unc-46(e177) mdf-1(gk2) h1992 unc-46(e177) mdf-1(gk2); h1988ts  252.8  ± ±  + ±  ±  8.2 (6)  297.7  ±  ±  ±  5.1 (6)  14.2 (5)*  7.2 (5)*  unc-46(e177) mdf-I(gk2); h1987  178.0  unc-46(e177) mdf-1(gk2); h1958  177.5+ 11.2(6)  138.7 ± 4.8 (6)  316.2  unc-46(e177) mdf-I(gk2) h1985  158.8  149.2  308.0 ± 8.3 (5)  ±  ±  8.1 (5)  8.1 (5)  ±  ±  3.5 (5)  6.6 (5)  Suppressor strains that carry ruIs32, an integrated H2B::GFP transgene (PRAITIS  ±  9.5 (6)  et al. 2001), were  constructed and analyzed for anaphase onset. Asterisks denote a significant difference as shown by p values of the student’s t-test statistic.  2.3.4  The Class II suppressor, h1985, displays an aberrant apoptotic response In mouse cells, it was shown that lethality associated with deletion of the Mad2 gene  can be suppressed by the deletion of p.53 to yield viable Mad2 p53 cells, which exhibit extraordinarily high level of CIN (BuRDs et a?. 2005). The authors showed that the deletion of p53 did not alter mitotic timing in Mad2 cells (BuRDs et a?. 2005). In support of these findings, WANG  et a?. (2004) showed that haploid loss of the p53 gene rescued the embryonic lethality in  mice caused by targeted deletion of Brcal, exon 11 (Brca1”’ 1) and animals developed into adults exhibiting increased tumorigenesis and chromosomal abnormalities (WANG et al. 2004). They further showed that Brcal controls the spindle checkpoint by regulating the expression of MAD2. These findings would suggest that lethality associated with loss of the mitotic checkpoint  47 is a result of chromosome breakages, fusions and other DNA damage that triggers apoptotic response. Therefore, it is possible that mutations that result in defective apoptosis could rescue the mdf4 lethality while having a normal anaphase onset. To investigate this, the suppressor mutants were analyzed for apoptotic response in the absence of MDF- 1. There are two waves of apoptotic cell deaths in C. elegans. The first occurs during the somatic development when 131 of the 1090 cells undergo programmed cell death in highly reproducible manner to generate an adult hermaphrodite that contains 959 nuclei outside the germ line (STERGI0u and HENGARTNER 2004). The second wave of programmed cell death occurs in the adult germ line, where approximately half of the female germ cells undergo developmentally programmed “physiological” cell death during oogenesis (STERGI0u and HENGAWrNER  2004). Unlike somatic cells, which have a fixed cell division program and silenced  checkpoint during the DNA damage response in embryos (H0LwAY et al. 2006), germline nuclei in C. elegans undergo checkpoint control and apoptosis in response to genotoxic stresses. Under normal growth conditions on average two apoptotic cells can be observed at any given time in an adult hermaphrodite gonad due to physiological cell death (STERGI0u and HENGARmER 2004; Figure 2.4B). Under genotoxic stress the number of apoptotic cell deaths is significantly increased (STERGI0u and HENGARmJER 2004). To monitor apoptosis in suppressor mutants, the vital die SYTOI2 was used, which specifically stains apoptotic bodies (GuMIENNY et al. 1999). In wild-type animals, an average of 1.60±0.15 (n80 gonad arms) SYTO12-stained corpses per gonad arm was observed (Figure 2.4). In F 1 mdf-1 animals, a significant, more than two-fold, increase in number of apoptotic corpses was observed that was further elevated in the F 2 mdJ4 homozygotes to 7.75±0.58 (n52 gonad arms) corpses per gonad arm (Figure 2.4). These data support the hypothesis that in the absence of the checkpoint DNA damage arises and acumulates triggering the apoptotic response. Furthermore, it was also observed that all of the Class I suppressors that delay anaphase onset display an elevated number of SYTO 12-stained  48  A  9  C 0 C’,  C’, 0. 0 C) 4-  0  a0  0. CC’  I 0  S(cV  B  h1985 mdf-1(gk2)  Figure 2.4: Apoptotic response in the suppressors (A) Quantification of SYTO12-stained cells in the gravid hermaphrodite gonads. Bars represent the mean number of corpses per gonad arm with SEM error bars. (B) The representative images of SYTO 12-stained wild-type, mdJJ(gk2) F 1 and mdfl(gk2) h1985 gonads. All the measurements were performed at 20°C.  corpses when compared to wild-type animals, decreased number of corpses when compared to F 2 mdf-1 animals, and not significantly different amount of apoptotic corpses than the F 1 mdJJ  animals (Figure 2.4). These results, in accordance to our previous data, suggest that the majority of the suppressors bypass the MDF- 1 checkpont requirement for viability, but they still display  49 high levels of CIN and DNA damage (TARAIL0 et al. 2007). Interestingly, in one of the Class II suppressors, h1985, an average of 1.26±0.17 (n=97) corpses per gonad arm was observed (Figure 2.4). These data suggest that at least one of the Class II suppressors might bypass the checkpoint requirement by altering the apoptotic response.  2.3.5  Neither cep-1 nor ced-3 rescue the mdf-1(gk2) lethality Next it was tested whether the defective apoptosis by itself could bypass the MDF-1  checkpoint requirement in C. elegans. Worm genes can be inactivated by feeding animals on bacteria expressing double-stranded RNA homologs to a specific worm gene. CEP-1 is the C. elegans p53 homolog that is required for DNA damage-induced apoptosis (DERRY et al. 2001). The cep-1 gene was efficiently inactivated in F 1 mdf-1 homozygotes using the RNAi by feeding method (Figure 2.5). Loss of CEP-1 reduced the number of apoptotic corpses observed in mdf-1 mutants (Figure 2.5). This indicates that the increased apoptosis observed in the absence of MDF-1 is dependent on the DNA-damage checkpoint protein CEP-1. However, loss of CEP-1 did not rescue the mdJ] lethality in C. elegans. To further investigate if complete loss of cell death (both programmed and DNA damage-induced) could rescue the mdf-1 lethality, the strain of the genotype: unc-46(el 77) mdJl(gk2) +/+  +  nTl [let-X]; ced-3(n7] 7) was constructed.  CED-3 is a member of caspase family of cystein proteases required for the killing process (STERGI0u  and HENGARTNER 2004). SYTO-12-staining of the mdJ1(gk2); ced-3(n71 7)  homozygotes confirmed that apoptosis was completely abolished in these animals (Figure 2.5); however, the absence of CED-3 did not bypass the MDF- 1 checkpoint requirement for survival as none of the mdf4(gk2); ced-3(n71 7) homozygotes could be maintained for longer than two generations. These data led to conclusion that although one of the suppressors displays a defective apoptosis phenotype, loss of apoptosis cannot rescue the mdJ4 (gk2) lethality in C. elegans.  50  A  B 4.5 4 3.5 3 25 2 1.5 1 05 0  Figure 2.5: Neither cep-1 nor ced-3 rescue the mdf-1 (gk2) lethality: (A) Quantification of SYTO 12stained cells in the gravid hermaphrodite gonads. Bars represent the mean number of corpses per gonad arm with SEM error bars. (B) The representative image of the SYTO12-stained mdJl(gk2); ced-3(n717) adult gonad. All the measurements were performed at 20°C.  51  2.4  DISCUSSION  The spindle assembly checkpoint prevents aneuploidy by delaying the metaphase-to anaphase transition until all chromosomes are successfully attached to microtubules at kinetochores. In the absence of MDF-1, chromosome mis-segregation arises leading to highly penetrant maternal effect lethality/sterility (KITAGAwA and RosE 1999). The results presented in this thesis show that the majority of the mutations that bypass the MDF-1/Madl checkpoint requirement for survival and fertility delay mitotic divisions in germline and early embryonic cells. In addition, a second class of suppressors has been identified that works through an  unknown mechanism, which neither delays anaphase onset nor accumulates securin. Normally, activation of the SAC in response to spindle defects delays mitotic division in the early embryo and germline (ENcALADA et al. 2005; KITAGAwA and ROSE 1999). The first demonstration that mitotic delay could compensate for the lack of checkpoint was that mutations in the emb-30/APC4 and fzy-1/CDC2O genes have the ability to suppress the mdf-] (gk2) lethal phenotype (FuRuTA et a?. 2000; KITAGAwA et al. 2002). This thesis reports on these and additional mutants that cause delayed mitosis, which correlates with securin accumulation. It is likely that the anaphase onset delays observed in these mutants allow more time for proper kinetochore attachment, thus preventing defects in chromosome segregation. If this were true, those suppressors with longer mitotic delays would be expected to improve chromosome segregation fidelity and result in better viability (Figure 2.6). In general this was observed. An increase in mitotic delay correlated with a decrease in X-chromosome missegregation and, to a lesser extent, with an increase in viability (Figure 2.6). For instance, h1960 suppressor delays mitosis from 304 (mdJl) to 629 seconds, decreases X-chromosome missegregation from 5% to 0.8% and improves viability from 2% to 42% (Table 2.1 and Figure 2.2). However, there are exceptions; for example, for the emb-30(tn377ts) h1959ts and h1962ts suppressor alleles, the ,  52  A  B  10  60  .  A  8  •  0  4O• 0  E  •  A  0  A A  2  A A  IL I  1.0  I  I  I  I  1.2 1.4 1.8 1.6 2.0 Mitotic timing (fold increase)  I  2.2  I  10  I  I  I  I  2 Mitotic timing (fold increase)  I  2  Figure 2.6: Correlating mitotic delay with survivability (%fertile progeny) and genome stability (Him).  (A) scatter plot of survivability (% fertile progeny; Table 2.1) and timing of mitotic division in early embryo (fold increase when compared to N2; Figure 2) and (B) genome stability (defined as % males; Table 2.1) and timing of mitotic division in early embryo (fold increase when compared to N2; Figure 2.2). mitotic delay correlates better with the reduction in X-chromosome missegregation than it does with the increase in viability (Table 2.1 and Figure 2.2). When removed from the mdf4 background, a wide range of viability reduction associated with the suppressor mutations was observed (Table 2.3). Thus, it is reasonable to speculate that the viability of the suppressor strains is affected by other consequences of a suppressor mutation in addition to mitotic delay. The cell cycle of early embryonic cells in C. elegans is rapid, consists entirely of S phase and mitosis, and lacks gap phases (Figure 1.1). The cell divisions in the early C. elegans embryo are asymmetric and asynchronous, which is crucial for normal development and cell fate  53 specification. It is unclear whether or not C. elegans embryos have functional S and M phase checkpoints. However, several lines of evidence suggest that the S phase checkpoint (atl-i and chk-]) is functional and developmentally controlled by preferential activation in the P1 blastomere to generate the 2-mm asynchrony (BRAucHLE et al. 2003). Attenuated asynchrony in chk-i(RNAi) and div-i mutants (DNA polymerase a) results in sterility and embryonic lethality, respectively (BRAucHLE et al. 2003; ENcALADA et a!. 2000). Recently, H0LwAY et al. (2006) showed that the S phase checkpoint is actively silenced during the DNA damage response in the early C. elegans embryo to ensure normal timing of cell division, even in the presence of heavily damaged chromosomes. In contrast, the duration of mitosis is consistent in early C. elegans embryonic cells (ENcALADA et al. 2000, ENcALADA et a!. 2005; TARAIL0 et a!. 2007) and the activated mitotic checkpoint will delay progression through mitosis in response to either chemical or mutational disruption of the embryonic microtubule cytoskeleton (ENcALADA et al. 2005). The average duration of these delays does not exceed 2.5-fold even after treatment with the microtubule destabilizing drug nocodazole (ENcALADA et a?. 2005). In accordance with this data, longer than 2.1-fold mitotic delays were not observed in the suppressors. Furthermore, the first two embryonic cell divisions in the suppressor mutants remained asynchronous. These observations suggest that an M phase delay is tolerated by C. elegans embryo better than an S phase delay. For instance, the hi960 suppressor delays progression through mitosis more than two-fold, yet it is viable (Table 2.3). The defects observed in h1960 embryos, as well as the rest of the suppressor mutations (Table 2.3), are mild compared to the embryonic lethality associated with S phase delays in the presence of replication problems (ENcALADA et a?. 2000; BRAucHLE et a?. 2003; H0LwAY et a?. 2006). These data suggest that the SAC might not be involved in developmental regulation contributing to the asynchrony of the early embryonic cells. Thus, the suppressors that delay mitotic progression might shed light on the relationship between S and M  54 phase checkpoints, the embryonic sensitivity to delayed interphase, and the embryonic tolerance to two-fold mitotic delays. Three suppressors, h1958, h1987, and h1985, rescue the mdf-1 lethality without delaying anaphase onset. The normal mitotic timing observed in these mutants suggests that these suppressors rescue the lethality by an alternate mechanism. Indeed, we showed that these suppressors do not accumulate securin, further suggesting normal APC/C activity. Furthermore, in contrast to the Class I suppressors, the h1958 and h1987 suppressors did not display Him phenotype when analyzed in the wild-type background (Table 2.3). In addition, the h1958 is the only suppressor that exhibits developmental delay phenotype (Table 2.3). An alternative mechanism known to render the spindle checkpoint nonessential for survival was described in mouse cells. BuRDs et al. (2005) showed that inactivation of the p53 gene suppresses the lethality associated with deletion of the Mad2 gene without altering mitotic timing in Mad2 cells (BuRDS et al. 2005). Although apoptotic response was found to be altered in one of the three Class II suppressors, h1985, it is unlikely that loss of apoptosis rescues the mdJ](gk2) lethality since loss of function or absence of the ced-3 and cep-] genes could not rescue the mdf 1 (gk2) lethality in C. elegans. These results suggest that in contrast to murine cells, the elevated apoptosis triggered by damage in the absence of the spindle checkpoint cannot account for the lethality observed in the mdf-1(gk2) homozygotes in C. elegans. Thus, the Class II suppressors represent unexplored functions that render SAC nonessential for long-term survival and fertility in C. elegans. In conclusion, using time-lapse imaging of early embryonic cells and germline mitotic division, two classes of suppressors were described. The Class I suppressors rescue the mdfi 1 (gk2) lethality by constitutively delaying mitotic divisions. The Class II suppressors bypass the MDF-l checkpoint requirement by an unknown mechanism. When separated from mdf-1(gk2) the suppressors are viable, but majority exhibits chromosomal abnormalities and variable  55 reductions in fitness. Despite rescuing the viability of animals in the absence of the MDF-1 checkpoint, the suppressors still display high levels of chromosomal instability. Thus, in addition to increasing our understanding of the regulation of chromosome segregation and early embryonic development this collection of mutants could also provide us with new valuable insights into cancer development.  56  2.5  BRAucHLE,  BIBLIOGRAPHY  M., K. BAuMER and P. GoNczY, 2003 Differential activation of the DNA replication  checkpoint contributes to asynchrony of cell division in C. elegans embryos. Cuff. Biol. 13: 819-827. BRENNER, BuRDs,  S., 1974 The genetics of Caenorhabditis elegans. Genetics 77: 71-94.  A. A., A. S. LuTuM and P. K. S0RGER, 2005 Generating chromosome instability through the simultaneous deletion of Mad2 and p53. PNAS 102: 11296-11301.  CHEuNG,  I., M. ScHERTzER, A. M. ROSE and P. M. LANsD0RP, 2002 Disruption of dog-i in  Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA. Nat. Genet.  31: 405-409. DERRY,  W. B., P. A. PuTzKE, and J. H. R0THMAN, 2001 Caenorhabditis elegans p : 5 3 Role in Apoptosis, Meiosis and Stress Resistance. Science 294: 591  ENcALADA,  —  595.  S.E., J. WILLIs, R. LYczAK and B. B0wERMAN, 2005 A spindle checkpoint functions  during mitosis in the early Caenorhabditis elegans embryo. Mol. Biol. Cell. 16: 10561070. ENCALADA,  S.E., P. R. MARTIN, J. P. PHILLIPs, R. LYczAK, D. R. HAMILL et al., 2000 DNA  replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos. Dev. Biol. 228: 225-238. FuRuTA,  T., S. TucK, J. KIRcI-i1’JER, B. KOCH, R. AuTY, R et al., 2000 EMB-30: an APC-4  homologue required for metaphase to anaphase transition during meiosis and mitosis in Caenorhabditis elegans. Mol. Biol. Cell. 11: 1401-1419. GuMIENNY,  T. L., E. LAMBIE, E. HARTwIEG, H. R. HoRvrrz, and M. 0. HENGARTNER, 1999  Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline. Development 126: 1011-1022.  57 HoDGKni,  J. A., H. R. HoRvITz and S. BRENNER, 1979 Nondisjunction mutants of the nematode  C. elegans. Genetics 91: 67-94. A. H., S. H. KIM, A. LAV0LPE and W. M. MIcHAEL, 2006 Checkpoint silencing during  H0LwAY,  the DNA damage response in Caenorhabditis elegans embryos. 3. Cell Biol. 172: 9991008. KITAGAwA,  R., and A. M. ROSE, 1999 Components of the spindle-assembly checkpoint are  essential in Caenorhabditis elegans. Nat. Cell Biol. 1: 514-521. KITAGAwA,  R., E. LAw, L. TANG and A. M. RosE, 2002 The Cdc2O homolog, FZY-1, and its  interacting protein, IFY- 1, are required for proper chromosome segregation in Caenorhabditis elegans. Cuff. Biol. 12: 2118-2123. Li, R., and A. W. MuRRAY, 1991 Feedback control of mitosis in budding yeast. Cell 66: 519-531. PRAITIS,  V., E. CASEY, D. COLLAR and 3. AuSTIN, 2001 Creation of low-copy integrated  transgenic lines in Caenorhabditis elegans. Genetics 157: 1217-1226. RosE, A. M., and D. L. BAILLIE, 1979 Effect of temperature and parental age on recombination and nondisjunction in Caenorhabditis elegans. Genetics 92: 409-418. STERGIOu,  L., and M. 0. HENGAwrNER, 2004 Death and more: DNA damage response pathways  in the nematode C. elegans. Nat. Cell Death and Diff. 11: 2 1-28. TARAILO,  M., R. KITAGAwA and A. M. RoSE, 2007 Suppressors of spindle checkpoint defect  (such) mutants identify new mdf-]/MADJ interactors in Caenorhabditis elegans. Genetics 175: 1665 WANG,  —  1679.  Y., and J. D. BURKE, 1995 Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 15: 6838-6844.  WANG,  R. H., H. Yu and C. X. DENG, 2004 A requirement for breast-cancer-associated gene 1 (BRCA1) in the spindle checkpoint. PNAS 101: 17108-17113.  58  Chapter 3’: Characterization and Mapping of the Suppressors 3.1  INTRODUCTION  The target of the spindle assembly checkpoint (SAC) and major regulator of the timing of anaphase onset is the anaphase promoting complex/cyclosome (APCIC), a large conserved 1 .5-MDa E3 ubiquitin ligase complex consisting of 10-15 subunits, depending on the species (reviewed in PETERS 2006; Table 1.2). During the M phase, the activity of the APC/C is tightly regulated by the WD4O repeat protein Cdc2O/Fizzy to prevent chromosome mis-segregation (VIsINTIN et al. 1997). To date, 11 orthologs of the APC/C subunits have been identified in C. elegans (GOLDEN et al. 2000; DAvis at al. 2002; SHAKES et al. 2003; DONG at al. 2007; Table 1.2), including the APC/C co-activatorfzy-i/CDC2O (KITAGAwA et al. 2002). Previously, it was shown that the reduction of activity in the APC/C component, emb-30(tn377ts)/APC4, and its activator fzy-i (hi 983)/CDC2O, can suppress the lethality and sterility associated with the absence of MDF-1 (FuRuTA et al. 2000; KITAGAwA et al. 2002). Recently, STEII’J et al. (2007) isolated mdf-i(avi9) allele in a screen for suppressors of the mat-3(ori8O)/CDC23/APC8 lethality. This group also showed that the mdJi(av19) allele does not exhibit APC/C subunit specificity since it was able to suppress at least one mutant allele of each APC/C subunit mutants identified to date (STEIN et al. 2007). Having established that the majority of the suppressors of the mdJi (gk2) lethality, isolated in our screen, might bypass the checkpoint requirement by reducing the APC/C activity, which results in securin accumulation and delayed anaphase onset (TARAILO  1  et al. 2007), it was reasonable to speculate that some of the suppressors may represent  A version of this chapter has been published: M., R. KITAGAWA and A. M. RosE, 2007 Suppressors of spindle checkpoint defect (such) mutants identify new mdf-i/MADJ interactors in Caenorhabditis elegans. Genetics 175: 1665 1679.  TARAIL0,  —  59 known or novel downstream targets of the SAC. In this thesis, all of the suppressors of the mdf 1(gk2) lethality were analyzed for their molecular basis and additional phenotypic consequences of the suppressor lesions.  60  3.2  MATERIALS AND METHODS  The source of mutations is given in Chapter 2: MATERIALS and METHODS.  3.2.1  C. elegans strains, alleles and culturing The Bristol strain N2 was used as the standard wild-type strain (BRENNER 1974). The  marker mutations and balancer chromosomes used are listed in the order of chromosomes. LGI: dpy-5(e6]), dpy-5(s]300); LGII: dpy-1O(e128), unc-4(e]20); LGIII: dpy-1 7(e164), dpy 18(e364), unc-32(e289) and LGIV: dpy-13(e]84) and LGV: unc-46(e177), mdf-1(gk2) and nTl(IV,V). The strains used include: CB4856, VC147 apc-]O(gk143), KR3627 unc-46(e177) mdf](gk2) +/+ + nT] [let-X] and DG627 emb-30(tn377ts). The CB4856 strain was used for single nucleotide polymorphism (SNP) mapping as described by WICKS et al. (2001). The strains were obtained from the Caenorhabditis Genetics Center. Animals were maintained using standard procedures (BRENNER 1974). 3.2.2  Genetic mapping using visible markers The such mutations were mapped to a chromosome using phenotypic Dpy (Dumpy)  markers located in the central regions of different autosomes. The suppressor containing hermaphrodites were mated to the dpy heterozygous males and the hermaphrodites of the genotype such/+; +/dpy; mdf-] unc-46/+ + were obtained and let to self. The DpyUnc-46 animals segregated from these heterozygotes were collected and observed for the presence of the suppressed mdf-1 (gk2) phenotype. If the suppressor is located on the same chromosome as dpy mutation then the DpyUnc-46Mdf- 1 animals would obtain a suppressor mutation only in the case of recombination event, less that 25% of the time.  61 3.2.3  Genetic mapping using snip-SNP markers The CB4856 strain, an isolate from a Hawaiian island, shows uniformly high density of  polymorphisms compared with the reference Bristol N2 strain (WICKS et al. 2001). The rapid mapping of the suppressors can be achieved by crossing the CB4856 males into a suppressed unc-46 mdf-1 hermaphrodites and generating progeny heterozygous for all markers. The Unc 46Mdf-1 progeny segregated from a heterozygote were individually plated and analyzed for the suppression. Two bulked lysates were formed, as described (WIcKs et al. 2001). One contained 100 suppressed “mutant” worms and the other contained unsuppressed “wild-type” worms. The proximity of a linked marker to a suppressor was examined by the relative proportion of each form of the biallelic marker in populations of “mutant” and “wild-type” lysates. 3.2.4  Sequencing The candidate genes located in the mapped regions were sequenced. The candidate  genes, including 300bp upstream and downstream, were PCR-amplified from genomic DNA isolated from the suppressor strains, purified and submitted for sequencing (Nucleic Acid and Protein Services, UBC). In the case of the mat-2 candidate gene, the full-length cDNA from the h]989 and h1987 suppressors was amplified using reverse transcription-PCR (RT-PCR) and sequenced (Nucleic Acid and Protein Services, UBC). 3.2.5  DAPI staining Animals were shifted at L4 stage from 20°C to 25°C and adult progeny were prepared  for whole-mount DAPI staining. One day old synchronized adults were washed with M9 buffer and stained with 1 5OnM DAPI in ethanol for 90 minutes at room temperature. Animals were destained overnight in M9 buffer at 4°C. Destained animals were mounted on agarose pads and viewed with the Zeiss Axioscope fluorescent microscope with 40X objective. A Retiga 2000R camera (Qimaging) and Openlab 4.0.2 software (Improvision) were used to acquire images.  62 3.2.6  Identification of the h1988ts, h1959ts and h1962ts suppressors The h1988ts suppressor was positioned using snip-SNPs between C34F1 1 and K03H9,  where fzy-1 is located. The fzy-1 gene, including 300bp upstream and downstream, was PCR amplified from the h1988ts strain and sequenced (Nucleic Acid and Protein Services, UBC). The hi9S9ts and h1962ts suppressors were assigned using snip-SNPs to a region where apc-2 and emb-30 genes are located. The complementation analysis was performed by mating emb 30(h1959ts) males to unc-32(e289) emb-30(tn377ts) hermaphrodites at 20°C and shifting  +  emb  30(h1959ts)/unc-32(e289) emb-30(tn377ts) progeny at L4 stage to 25°C and by mating dpy 1 7(e164) emb-30(h1962ts) males to unc-32(e289) emb-30(tn377ts) hermaphrodites at 20°C and shifting dpy-1 7(e164)  +  emb-30(h1962ts)/+ unc-32(e289) emb-30(tn377ts) progeny at L4 stage  to 25°C. The full-length cDNA of the emb-30/APC4 gene from the h1959ts and h1962ts suppressors was RT-PCR amplified and sequenced (Nucleic Acid and Protein Services, UBC). 3.2.7  Identification of the such-i (h1960) suppressor such-i(h1960) was positioned using dpy-17 and dpy-18 markers and snip-SNP  mapping procedures between T28D6 and Y41C4A, a small region containing 40 predicted genes. The full-length eDNA of the Y66D 1 2A. 17 gene was amplified from both the such-i (hi 960) and N2 strains using RT-PCR and sequenced (Nucleic Acid and Protein Services, UBC). The G8OA change present in the such-i (hi960) strain was not observed in N2. This analysis also revealed that the RT-PCR product is shorter than the predicted cDNA (WormBase WS 164) in both N2 and such-i (hi960) strains. Sequencing revealed that 96bp of the exon III (from 591 to 686) predicted to encode for a protein are spliced out and as a result of this, SUCH-i is 32 amino acids shorter than predicted in both N2 and such-i (h1960). 3.2.8  RNA interference Individual bacterial RNAi feeding strains (RNAi control and gfi-3) were prepared as  described in Chapter 2: MATERIALS and METHODS. Twenty L4 such-i (h1960) and 20 wild-  63 type (N2) hermaphrodites were plated on each of these plates and fed dsRNA-expressing bacteria (RNAi control and gfl-3). Half of the plates were grown at 20°C and half at 25°C. 3.2.9  The UV response and SYTO12 Staining Staged young adult worms (12h post the L4 stage) were exposed to 2 100J/m of UV.  12h post treatment adult hermaphrodites were stained with SYTO12 as described in Chapter 2: MATERIALS and METHODS.  64  3.3  3.3.1  RESULTS  Three Class I suppressors are known APCIC components To genetically map the suppressors, phenotypic markers in combination with the snip  SNP (single nucleotide polymorphism) mapping procedure have been used as described by WICKS  et al. (2001) (see MATERIALs and METHoDs). The mapping data positioned three  suppressors to chromosome V, four to chromosome III and three to the gene cluster of chromosome II (Table 3.1). The cell cycle timing analysis showed that the majority of suppressors delay anaphase onset (TARAIL0 et al. 2007), which prompted us to examine if any of the suppressors represent known downstream targets of the SAC. 3.3.1.1  The h1988ts suppressor is a new allele offzy-1/CDC2O  The Class I suppressor, h1988ts, was mapped to the gene cluster of chromosome II, where the known SAC effector and mdf-] suppressorfzy-] is located. Sequencing offzy-] from the h1988ts suppressor strain identified a single base pair change from guanine to adenine at position 1,133 of the cDNA sequence in exon IX. This change caused an R378Q alteration in the amino acid sequence in the nonconserved region within the WD4O domain (Table 3.1). The fzy ](h1988ts) allele is more severe than the previously identified fzy-](h1983) allele (KITAGAwA et al. 2002). fzy-1(h]988ts) homozygotes can be maintained at 20°C but display severely reduced brood size (22 progeny) of which 49% arrest as embryos, 20% arrest as larvae and 31% develop into adults. The strain also has a high incidence of males (Him) phenotype (5% of the adult progeny are males). At 25°C the fzy-](h1988ts) homozygotes are 100% sterile. 3.3.1.2  The h1959ts and h1962ts suppressors are new alleles of emb-30/APC4  The Class I suppressors, h]959ts and h]962ts, were positioned in the gene cluster on chromosome III, where the known APC/C components apc-2 and emb-30 are located. These suppressor alleles are temperature-sensitive and cannot be maintained at 25°C.  65 TABLE 3.1 Positional cloning of such genes Alleles of such genes  Mapping and sequencing data  LG II fzy-1(h19881s)  R378Q in WD4O domain of FZY-l.  such-3(h 1989)  Located in gene cluster on II. No sequence  such-S(h1987)  changes in the scc-I, fj’-I,fzy-I, emb-27 genes or the mat-2 cDNA.  LG III emb-30 (hI9S9ts)  M4801 in nonconserved region of EMB-30.  emb-30 (h1962ts)  T2651 in WD4O domain of EMB-30.  such-I (h1960)  G27D in semiconserved region ofY66DI2A.17.  such-6 (h1958)  Closely linked to dpy-I 7. No sequence changes in the apc-1 I coding region.  LG V such-2(h1992)  Located in gene cluster on V. Not duplicated mdf I as determined by PCR and RT-PCR. No sequence changes in the apc-1O coding region.  such-4(h2168)  Maps to the right of unc-46. Not duplicated mdJ4 as determined by PCR and RT-PCR.  such-7(h1985)  Maps to the right of unc-46. Not duplicated mdf-l as determined by PCR and RT-PCR.  The map position, delayed mitosis and temperature sensitive phenotype of the h]959ts and h1962ts suppressors suggested that these might be new alleles of the emb-30 gene. Both of the suppressors failed to complement emb-30(tn377ts). Sequencing of emb-30 from the h1959ts  66 suppressor strain identified a single base pair change from guanine to adenine at position 1,440 of the eDNA sequence in exon VII. This change caused an M4801 alteration in the amino acid sequence within the nonconserved region. Similarly, sequencing of the h1962ts suppressor identified a single base pair change from cytosine to thymine at position 796 of the cDNA sequence in exon V. This change caused a T2651 alteration in the amino acid sequence in the nonconserved region within the WD6 domain. The emb-30(h]959ts) allele is a new conditional allele that belongs to the same class as the previously identified suppressor allele emb 30(tn377ts) (FuRuTA et al. 2000). The emb-30(h]959ts) homozygotes are easily maintained at 20°C; the brood size is normal (237 progeny) with 18% embryonic arrest, 2% larval arrest, 80% progeny that develop into adults, of which 0.8% are males. At 25°C the emb-30(h1959ts) homozygotes are 100% embryonic lethal. Interestingly, the second emb-30(h1962ts) allele does not belong to any of the previously described classes (FuRuTA et al. 2000). 3.3.1.3  The sterility of the emb-30(h19621s) allele is specific to sperm  The dpy-] 7(e164) emb-30(h1962ts) worms displayed a characteristic phenotype at restrictive temperature as the majority of the brood at 25°C consisted of unfertilized oocytes (Table 3.2). The emb-30(h1962ts) phenotype was further analyzed in the absence of the dpy 17(e164) background and it was observed that emb-30(h1962ts) worms laid mainly unfertilized oocytes at both permissive and restrictive temperatures (Table 3.2). The emb-30(h1962ts) homozygotes can be maintained at 20°C, as all of the adult progeny (23%) segregated from the emb-30(h1962ts) mothers cultured at 20°C produced ‘30% fertilized oocytes (Table 3.2). However, at 25°C all of the adult progeny (18%) segregated from the emb-30(h1962ts) mothers laid 100% unfertilized oocytes (Table 3.2). In the emb-30(h1962ts) broods at both permissive and restrictive temperatures a higher incidence of males and some embryonic and larval arrest were observed (Table 3.2). These observations suggest that the emb-30(h1962ts) allele is a new conditional allele that results in sterility at 25°C due to unfertilized oocytes.  67 TABLE 3.2 The emb-30(h1962ts) mutant phenotype Parental genotype  Broods  Unfertilized Embryonic  Larval  Adult  Males  size  oocytes (%)  arrest (%)  arrest (%)  (%)  (%)  dpy-17(e164) emb-30(h1962ts)  49  74.5  7.1  1.2  17.2  2.2  dpy-17(e164) emb-30(h1962ts)XN2c3’  56  0  0  0  100  50.8  dpy-17(e164) control  139  37.2  0  0  62.8  0.3  emb-30(h1962ts)  74  73.5  6.7  2.2  17.6  3.3  emb-30(h1962ts)XN2c3’  111  1.8  1.2  1.4  95.6  49.1  dpy-17(e164) emb-30(h1962ts)  117  35.2  2.4  4.1  58.3  1.0  dpy-17(e164) emb-30(h1962ts)XN2c3’  170  0.6  2.8  1.2  95.4  50.0  dpy-17(e164) control  244  26.7  0  0  73.3  0.1  emb-30(h1962ts)  285  70.3  4.3  1.9  23.5  1.3  emb-30(h1962ts)XN2c3’  243  17.0  1.5  0.9  80.6  45.7  Temperature 25°C  Temperature 20°C  ‘Brood size is defined as a total number of shelled eggs and unfertilized oocytes laid by a single hermaphrodite.  Many mutations that eliminate spermatogenesis or cause production of defective sperm in hermaphrodites are recognized by unfertilized, round, brown oocytes that lack an eggshell, resulting in self-sterile hermaphrodites (reviewed in L’HERNAuLT 2006). To analyze the nature of the sterility in the emb-30(h1962ts) hermaphrodites, several L4 emb-30(h1962ts) hermaphrodites were mated to wild-type males at both restrictive and permissive temperatures. If these hermaphrodites had defective spermatogenesis, their oocytes would have been fertilized by wild-type male sperm. Wild-type sperm were capable of fertilizing emb-30(h1962ts) oocytes at  68 both temperatures and in all strains tested (Table 3.2). Moreover, the majority of the broods consisted of fertilized oocytes that developed into fertile adults (Table 3.2). In contrast, the wildtype sperm did not rescue the tn377ts and h1959ts alleles of the emb-30 gene, which result in an embryonic arrest phenotype at restrictive temperature. These data led to conclusion that the sterility observed in the emb-30(h1962ts) mutant is a consequence of defective spermatogenesis. To analyze whether emb-30(h]962ts) hermaphrodites produce defective sperm incapable of fertilization or a limited amount or no sperm at restrictive temperature, one day-old adult progeny from emb-30(h1962ts) mothers at 25°C were fixed and stained with DAPI (Figure 3.1). If the emb-30(h1962ts) hermaphrodites produced sperm incapable of fertilization, it would be expected that many sperm accumulated in the spermatheca. On the contrary, all of the examined emb-30(h1962ts) hermaphrodites segregated from mothers cultured at 25°C (20/20) contained no detectable sperm (Figures 3.1B and 3.1D). It was also observed that emb 30(h1962ts) oocytes pass through spermatheca, resume meiosis, continue cycling and become highly polyploid, endomitotic oocytes (Emo) (Figure 3.1). The Emo phenotype is common to ovulated but unfertilized oocytes in C. elegans (McCARTER et al. 1999). These results provide further evidence that emb-30(h]962ts) hermaphrodites produce normal oocytes and defective sperm.  3.3.2  The such genes identify new mdf-1IM4DJ interactors Seven suppressor mutations do not appear to be alleles of known C. elegans  metaphase-to-anaphase transition pathway components and were named such (for suppressor of spindle checkpoint defect): such-i (h1960), such-2(h1992), such-3(h1989), such-4(h2168), such 5(h1987), such-6(h1958) and such-7(h1985) (Table 3.1).  69  Wild-type  emb-30(h I 962ts)  Figure 3.1: The emb-30(h1962ts) allele has an Emo phenotype at 25°C. Animals were shifted in LA stage from 20°C to 25°C and their adult progeny were DAPI stained as young adults. Images show partial gonad and partial uterus (A and B), or the entire uterus (C and D). In wild-type hermaphrodites (A and C) the highly condensed haploid sperm nuclei can be seen in the spermatheca (white arrowhead). Left of the spermatheca, embryos are located within the uterus (A and C). In emb-30(h1962ts) animals (B and D), there is no sperm DNA detected in the spermatheca (arrowhead) and the uterus is filled with Emo oocytes only (arrows). (E) Schematic diagram of a single wild-type adult hermaphrodite gonad arm including half of the uterus. A maturing oocyte is pushed into the spermatheca, successfully fertilized by sperm and pushed into the uterus where it develops into embryo. (F) Schematic diagram of single emb-30(h1962ts) adult hermaphrodite gonad arm including half of the uterus. A maturing oocyte is pushed into the spermatheca. The lack of sperm results in a failure to fertilize. The unfertilized oocytes pass through spermatheca into the uterus, continue cycling and become Emo, highly polyploidy, round, brown oocytes  that lack an eggshell. I am grateful to Marko Graovac for his help on preparation of the Figures E and F. Scale bars: 50 tim.  70 3.3.2.1  such-i (h1960) corresponds to Y66D12A.17, an APC5 —like gene  The such-] (h]960) was assigned to a region on the right arm of chromosome III, where no known components are located (Table 3.1). The h]960 allele has no obvious phenotypes in the wild-type background at 20°C; the brood size is normal (260 progeny) with 7% embryonic arrest, 93% progeny that develop into adults and 0.2% males. The such-] homozygotes are also viable at 25°C, but higher temperature intensified the phenotype as the brood size decreased (172 progeny), embryonic arrest increased to 46% and only 39% of progeny developed into adults. It was also observed that such-] has a Him phenotype (3.6%) at 25°C. The such-] suppressor was positioned using snip-SNP mapping between T28D6 and Y41C4A, a small region containing 40 predicted genes (Figure 3.2A). One of the genes within this region is Y66D 1 2A. 17, a previously unknown gene that appears to be similar to the human anaphase promoting complex subunit APC5 (WormBase WS164). The such-] (h]960) suppressor displays a delayed mitosis phenotype in both germline and early embryonic cells as a result of delayed anaphase onset (Figures 2.1 and 2.2; and Table 2.4). This phenotype would have been expected as a consequence of reduced APC/C activity. Sequence analysis of the coding region of Y66D12A.17 from the such-] (h1960) strain revealed a single base pair change from guanine to adenine at position 80 of the eDNA sequence in exon I. This change caused a G27D alteration in the amino acid sequence in the semiconserved region (Table 3.1 and Figure 3 .2B). Therefore, the Y66D12A.17 was renamed to such-] (WormBase WS179). There are ten APC/C subunits identified to date in C. elegans; some were isolated in forward genetic screens for ts emb mutants (CAssADA et al. 1981) and ts mat mutants (GoLDEN et al. 2000; SHAKES et al. 2003) and some were identified through sequence homologies (DAvIs et al. 2002; D0NG et al. 2007). gfi-3/M163.4 was reported to encode APC-5 (ZAcHARIAE et a!. 1998) and to share 11% identity and 20% similarity with S. cerevisiae Apc5p (DAVIS et a!.  __  71  A LG Ill  T28D6  Y66A7AL Y47D3B  Y66A7AR  Y66D12A  Y4IC4A  ZN Y66D12A.17  =  such4  B G2fl33h3960) • rt  964t112A. 17 GF3IAV11$ HAP9$  QKAPRPIX$YLDAQEVF9ELr1j 5V34ESLYPNPfl(V3UU3V2’Q- 2E11  Y441132A, 17 3/$C5  146111 2A 7 !A3C5 H$AC5  3  LKPPt)KDk4V PVSP SPH IN  LZIS3YD P  3DQI EMIP Md2PrLNV NEQVPOII  166012k. 17 0I- 3/%-t$  IRThV14AH 13.tL3 11XSRt3  .‘‘  ‘1  RJ1tLGRSXYYRFVTS  oGpDIn.s3cLy  8CPQVQS$X3UJ  SWI.DEL3SNSPLY1WIKRV)q4 169 179 PHKTSVVGLF--- JrLAYSILSPsQVFKtft’AI4 160  3661112A. 17 OFO -1/APCS  OLRLVOI.CP S%F1KNPR OASLLK3IDET  Y6611127. 19 3 1AP95 I4OAPC$  3MLTSKRALE1’LKLI(SPS SLSMPEL433VMAA)c114RLAVSTQ? 11813 LSKSNiG(JRS  364133 2A. I 7 11PI•3/6pS  V3 I 65  I  LAW 124 G 119 5Ua2 197  pxDpKL?ANorv)4goLMGD21opssNNs?Nymo4xku4  •RTSNGtPE1A!QKflSGTh9ZX$V2LSG1NRMpTEI TESr$KVPQ5NAKSVL$AVESK1HPEPXG6Y - IPHPI  3  AVPS S$ NRA? 62 VSUE $6  YPL04CE YYNUt?RSVKRH tPASUflZJPN  I cAA 224 ---V. $IN’JM 1132 P1.SQK EFFISQ 219  2PVQLSVKSLEQ IVOVOLK $55 )CAMKS YULQDVPSS7I1SLL11  !OPST.QThVNR APSIOARZ ThA1J6LM11  I  1188 292 276  LF6SIQ11S 344 LI L4SL®4 192 332  RI? !Vfl8SThSGPtLQRtS06?PAKXUES QMANIETrnLGCRAL18 DAKNZ IQH11LSW3jVO”-----------. --SDSYVL  NSVRAàV 494 RKALLHI8 495 VHGLF 334  I 661)1 2?. 17 (IF! 3/APCS  4VV3’L9 l,IPRDYBU8APLVSNCKFL GKPKNNSTADYVSVGPMRL 443 Q. 1N6HTR6GPcc11sSDPELVASYGEMU8U4Kt !AEG NYK11T1DRVAEGTTC 462 ---------- 5 416  366111 flkII (WI I 1?PI3 NAPC5  $LL1L9WGFVLQAFAMAXNF$G1IQ84YMRvA5194IV  -  LIST 519 PPVIPFQA $211  ----------  ?661112, 17  3  AGVI!VYS  YE  IG1W4SYS CN3EI  UOAPC$  53 V  3661112L 13 GP3fVC5  LN4WCSRNLAGLs-rsAp  HJPC5  !G46LAOSLV?GIThUSr  Y661112A.17  $IIQIQ  G6Z-3MPC5  P1SP RT  -  IDIUR ! SVUOIUIER  81 VT S  EAV’  SAN  463  ‘P1 579 P11 583 591  KIAEAVUaQIYSCGIIVRGS GEERASYr LNW533RGR  6?lX3OSEVLNt),Ai4SE TINThYOOP  1NWNAARHVDI 5$KQSNIUV1?I —QMAQ  ----14 434  LVNCQ3CLIO8TEI4V  I 637 Sal  102  KVAVNAflPL$ANI4?1VRRVOSLNLOR 6.3689 694 3*6OTGCLYThSOEPR 36PAt LSI LKVVXRRGAThIN4GQYV8CL 693 SI .SVAE3IYWRSSS P36 tJk1SKRLQYL5S86L4VA0LI5SPVQAI 641  3661112A17 1151-3/APIS HAP$4  0  ISA  S  15  76411126.17 1151 -3/APIS  914110  111- SF1 C 1  14.  NKWPANEKI.LLT5IT1 ArrKLVLlIEIAt CRISDWYPQ7LR  193436 C9’IXT 34PI  R1Y3131--!R1L116--VASAAS  9------8-- ----KKAEALEAA!  KOCKAQI 742 RALV 74$ EAEMYV 721  14S 1YPIEQKE4AKERPCK16ADPP P1 797 WV; IENRI667ICSFGKITADC39IRCEWW.5 800 1871 K3QSPPRCN4LFPQLI4QELPSHGVP4.S 341- 751  Figure 3.2: Molecular cloning and DNA sequence analysis of the such-1/Y66D12A.17 gene. (A) Mapping and cloning of the such-i (h1960) suppressor. such-i was positioned using snip-SNP mapping procedures between T28D6 and Y41C4A. (B) The Y66D12A.17 gene product GFI-3/Apc5 and Ii sapiens Apc5 proteins were aligned using ClustaIW. Common residues are shaded in black and the asterisk denotes the amino acid residue substituted in such-i (h1960).  72 2002). The RT-PCR analysis of the such-i gene from both the wild-type (N2) and h1960 strains revealed that 96 base pairs of the exon III predicted to encode for a protein (WormBase WS 164) are spliced out (TARAIL0 et al. 2007). As a result of this, SUCH-i is 32 amino acids shorter than predicted (Figure 3.2B). When the expressed SUCH-i was aligned to S. cerevisiae Apc5p, the 11% identity and 20% similarity was also observed. This would suggest that C. elegans has two APC5-like genes. The GFI-3 and SUCH-i are 37% identical and 57% similar (TARAIL0 et al. 2007). The GFI-3 and SUCH-i were then aligned to H sapiens homolog of APC5. While GFI-3 is 19% identical and 39% similar, the SUCH-i is 19% identical and 40% similar to the H sapiens APC5 (Figure 3.2B). These data further indicate that SUCH-i is a paralog of GH-3 and that both genes are Apc5p homologs in C. elegans. The recovery of the such-I (h1960) allele in this suppressor screen underscores the usefulness of the screen for identifying new components of the metaphase-to-anaphase transition pathway. 3.3.2.2  M163.4IAPC5 and F15H1O.3IAPC1O have paralogs in C. elegans  genome All of the subunits, identified in C. elegans, when inactivated by dsRNA resulted in embryonic lethality at the meiotic one-cell stage, with the exception of gfi-3!M163.4 and apc ]0!F15HIO.3, which encountered problems later in development (DAvIs at a!. 2002). The authors proposed that the C. elegans gJi-3/APC5 and apc-iO/APCJO components do not function as a part of the meiotic APC!C (DAvIs at al. 2002). The analysis of the mutants that rescue the mdfi(gk2) lethality suggests that the C. elegans genome has two functional copies of APC5; gIl 3/M163.4, which was previously described (ZAcHARIAE at a!. 1998; DAVIs at a!. 2002) and such-i! Y66D12A.17 described here. To analyze the consequence of the simultaneous inactivation of the gfi-3/M 163.4 and the such-i / Y66D 1 2A. 17 genes, the such-i (hi 960) animals were fed dsRNA-expressing bacteria for the gfi-3!M163.4 and analyzed at 20°C and 25°C. While the depletion of the gfl-3/M163.4 had no obvious effect on such-i (h1960) animals developing at  73 20°C when compared to the control, the depletion of the gfl-3/M163.4 gene product resulted in 100% of such-i (h1960) homozygotes arresting as embryos at 25°C (Figure 3.3A). In order to determine a stage of the embryonic arrests observed, the one-day old such-i (h1960); gfi-3(RNA1) adult hermaphrodites from 25°C were fixed and stained with DAPI (Figure 3.3 B). All of the examined such-i(h1960); gJI-3(RNAz) hermaphrodites cultured at 25°C (24/24) had uteri filled with the embryos arrested in one-cell stage (Figure 3.3B). These data suggest that the gfI 3/M163.4 and the such-li Y66D12A.17 genes are functionally redundant for progression through meiosis. Furthermore, these data suggest that such-l(h1960) is likely a conditional null and that such-li Y66D12A.17 gene, like gfi-3/M163.4 gene, becomes essential in the absence of its paralog. The apc-lO/F15H1O.3 is the second known APC/C component that was suggested not to be functioning as a part of the meiotic APC/C (DAVIS at al. 2002). The sequence analysis of the apc-lO/F15H1O.3 gene revealed that this gene may have a paralog as well, the Y48G1C.12 gene located on chromosome I (TARAILO et al. 2007). The analysis of the knockout allele, apc lO(gk]43), revealed that the apc-JO1F15H1O.3 gene is not essential in C. elegans (Table 3.3). The apc-lO(gk143) homozygotes can be maintained indefinitely but, similar to the emb 30(h1962ts) homozygotes, display unfertilized oocytes phenotype (Table 3.3). However, due to the lack of either the Y48G1 C. 12 mutant or the RNAi construct, the consequence of the simultaneous inactivation of the apc-iO/F15H1O.3 and the Y48G1C.12 genes was not investigated. Overall, these data suggest that all of the APC/C components identified in C. elegans may play an important role in meiosis. 3.3.2.3  such-2(h1992)  Class I suppressor, such-2, was mapped to the gene cluster of chromosome V where the apc-iO is located. Sequencing of the such-2 revealed no lesions in the apc-iO gene.  74  A  100 9O  •Eritrnic rJLarI  8O .  70  a)  -J  50 40 30 20 10  cia  I  S S  B  such-I (h1960) CRNA1 25CC  Figure 3.3: Both gfl-31M163.4 and such-li Y66D12A.l are required for meiosis. (A) The phenotypic  analysis of the such-i (h1960) animals fed RNAi control (empty construct) and such-i (h1960) animals fed RNAi towards gfi-3/M163.4 gene at 20°C and 25°C. (B) The such-i (hi960) animals fed RNAi control (empty construct) and such-i (h1960) animals fed RNAi towards gfi-3/M163.4 gene were shifted in L4 stage from 20°C to 25°C and their adult progeny were DAPI stained as young adults. Images show half of the uterus. In all such-i (h1960) hermaphrodites (19/19) grown on control RNAi plates (A and C) the uteri filled with multiple cell-stage embryos were observed. In all such-i (h1960) hermaphrodites (24/24) grown on gfi-3 RNAi plates (B and D), uteri filled with embryos arrested at one cell-stage were observed. Scale bars: 50 jim.  75 TABLE 3.3 The apc-1O(gk143) mutant phenotype Brood’  Unfertilized  Embryonic  size  oocytes (%)  arrest (%)  apc-1O(gk143)  169  94.6  1.5  0.2  3.7  N2(wild-type)  174  1.1  1.4  1.1  97.4  apc-1O(gk143)  545  90.8  0.6  0.0  8.6  N2(wild-rype)  269  0.8  0.2  0.0  99.0  Parental genotype  Larval arrest  (%)  Adult  (%)  Temperature 25°C  Temperature 20°C  Brood size is defined as a total number of shelled eggs and unfertilized oocytes laid by a single 1 hennaphrodite.  The such-2 suppressor was successfully isolated from the mdfii (gk2) and analyzed in wild-type background. The such-2 homozygotes can be maintained at 20°C; the brood size is close to normal (204 progeny) with 1% embryonic arrest, 99% progeny that develop into adults and 0.2% males. The h1992 homozygotes can also be maintained at 25°C, but higher temperature significantly intensified the phenotype as the brood size decreased (76 progeny), embryonic arrest increased to 95%, and only 5% of progeny developed into adults. 3.3.2.4  such-3(h 1989)  Class I suppressor, such-3, was mapped to the gene cluster on chromosome II, where many known downstream targets of the SAC are located. However, sequencing of this suppressor strain revealed no nucleotide changes in the sec-i, i)5’-i, fzy-i, emb-2 7 and mat-2 candidates (Table 3.1). The such-3 suppressor could not be isolated from the mdfii(gk2) background.  76 3.3.2.5  such-4(h2168)  Class I suppressor, such-4, was positioned to the gene cluster of chromosome V. The such-4(h2168) complemented apc-1O(gk143) and was later assigned to the region where no known metaphase-to-anaphase transition pathway components are located (Table 3.1). such-4 could not be isolated from the mq’f 1 (gk2) background. such-4 was analyzed for deletions of polyguanine tracts on chromosome V by PCR, but no lesions were detected (see APPENDIx A). 3.3.2.6  such-S(h1987)  Class II suppressor, such-5, was assigned to the gene cluster on chromosome II, where many known downstream targets of the SAC are located. However, sequencing of the suppressor strain revealed no lesions in the scc-1, i5’-1,fzy-1, emb-27 and mat-2 candidates (Table 3.1). The such-S suppressor was successfully isolated from the mdf1 (gk2) background and analyzed for phenotypes. such-S homozygotes can be maintained at 20°C; the brood size is decreased (164 progeny) with 8% embryonic arrest, 91% progeny that develop into adults and 0.1% males. The h1987 homozygotes are also viable at 25°C, but higher temperature intensified the phenotype as the brood size decreased (88 progeny), embryonic arrest increased to 12%, 55% of progeny arrested as larvae and only 33% of progeny developed into adults. Surprisingly, the such-S mutation does not display Him phenotype. 3.3.2.7  such-6(h1958)  Class II suppressor, such-6, was positioned in the gene cluster of chromosome III, where apc-1 1 gene is located; however, sequencing revealed no nucleotide changes in the apc-1 1 gene (Table 3.1). such-6 mutant does not display Him phenotype in the wild-type background and can be maintained at 20°C; the brood size is decreased (172 progeny) with 11% embryonic arrest, 48% progeny that develop into adults. The such-6 homozygotes are also viable at 25°C, but display reduced brood size (95 progeny), embryonic arrest 7% and 40% of progeny develop  77 into adults. Interestingly, the such-6 suppressor displays a significant developmental delay at both temperatures. 3.3.2.8  The Class II suppressor, sucli-7(h1985), abrogates DNA damage-  induced apoptosis The analysis of the apoptosis in suppressor mutants revealed that only such-7(h1985) alleviated the DNA damage response observed in the mdf-1(gk2) mutant; mdJ1 such-7 double mutants had fewer apoptotic corpses than mdf 1 single mutant (Figure 2.4A). In order to determine whether the low apoptosis in such-7 suppressor is due to the loss of apoptotic response to DNA damage or whether such-7 rescued the DNA damage in mdJl, the mdf-1 such-7 animals were treated with 100J/m 2 of UV. In N2 and mdfi] animals, an average of 10.98±0.89 (n=42) and 9.44±0.8 1 (n58) apoptotic corpses per gonad arm was observed, which suggests normal DNA damage-induced apoptosis (Figure 3.4). It was also observed that the Class II suppressors, such-S and such-6, had normal DNA damage response checkpoint (DDR). However, in the such-7 mdf-1 animals an average of 2.08±0.26 (n120) corpses per gonad arm was observed. This result suggests that such-7 suppressor abrogates the apoptosis induced by DNA damage (Figure 3.4). Furthermore, the such- 7 suppressor was positioned in a region on chromosome V, where atl-1 gene is located. ATL-l is the C. elegans ATR (homolog that is required for DNA damage-induced cell cycle arrest and apoptosis (Figure 3.4A) (GARCIA-MUSE and B0ULT0N 2005). However, sequencing revealed no nucleotide changes in the ati-] candidate. Recently, VAN HAAFTEN  et al. (2006) identified the F43D2.l gene, located in the such-7 region, as a new  gene that is required for DNA damage-induced cell cycle arrest and apoptosis. However, sequencing of the such-7(h]985) strain revealed no nucleotide changes in this candidate. There are no other genes known to affect both apoptosis and checkpoint activity located in this region.  78  Thus, it is possible that the such-7 gene is distinct from previously described checkpoint and/or apoptotic functions.  A E 12 10 0  -  -  8-  U, U, C.  6-  I  0 U U  4-  C.  2  0  C.  0x  B  —  e  mdf-1  +  UV  mdf-1: such-6  +  UV  rndf-1 such-7  +  UV  Figure 3.4: The such-7 suppressor abrogates DNA damage-induced apoptosis. (A) Quantification of  SYTO 12-stained cells in the gravid hermaphrodite gonads. Bars represent the mean number of corpses per gonad arm with SEM error bars. (B) The representative images of SYTO 12-stained mdJl(gk2) exposed to 1003 UV, mdJ4(gk2); such-5(h1987) exposed to 1003 UV, mdJl(gk2); such-6(h1958)  exposed to 100J UV and mdJ](gk2); such-7(h1985) exposed to 100J UV adult gonads. All the measurements were performed at 20°C.  79  3.4  DISCUSSION  The MDF-1 checkpoint is essential for C. elegans development under normal conditions. In this thesis, the mutations that bypass the MDF-1/Madl checkpoint requirement for survival have been analyzed. The molecular lesions have been identified in four of these suppressors, which correspond to three genes. All of the four suppressors belong to the Class I suppressors that delay mitotic divisions and include suppressor alleles of the known SAC effectors, emb-30/APC4 and fzy-i/CDC2O and the newly identified such-1/APC5-like gene. Furthermore, the phenotypic analysis, mapping and sequencing data suggest that components of the APC/C are unlikely to be responsible for the Class II suppressors that works through an  unknown mechanism, which neither delays anaphase onset nor accumulates securin. Interestingly, one of the Class II mutants appears to be critical for DNA damage-induced apoptosis. Previous work has shown that the reduced activity of the SAC effectors, emb 30(tn3 77ts) and fry-i (h1983), can suppress the lethality and sterility associated with the absence of MDF- 1 (FuRuTA et al. 2000; KITAGAwA et al. 2002). In this study, three new alleles offzy-i and emb-30 were described. emb-30 has been well studied in C. elegans. There are 17 alleles of emb-30 grouped to five different classes based on their mutant phenotype (FuRuTA et a!. 2000). The emb-30(h1959ts) allele is a new conditional allele, which belongs to the same class as the previously identified suppressor allele emb-30(tn377ts) (FuRuTA et a!. 2000). However, the emb 30(h1962ts) allele identified in the suppressor screen does not belong to any of the previously described classes. It displays a spermatogenesis defect. Previous work by FuRuTA et al. (2000) described oocyte specific alleles in the emb-30 gene. However, oocytes in emb-30(h1962ts) animals appear normal and can be fertilized by wild-type sperm and develop into adult progeny. The h1962ts mutation responsible for the sperm defect is the first mutation to be reported in the  80 highly divergent WD6 domain of EMB-30 (FuRuTA et at. 2000). The results presented in this thesis show that a mutation in this domain bypasses the MDF-l checkpoint requirement and results in defective spermatogenesis. Three suppressors, such-2, -3, and -4, bypass the checkpoint requirement likely by reducing the APC/C activity, which results in securin accumulation and constitutive delay in anaphase onset. The mapping and sequencing data in this thesis suggest that these suppressors will identify genes not previously known to function in the metaphase-to-anaphase transition in C. elegans. There are several lines of evidence that support this. First, three cloned Class I suppressors are mutations in emb-30 component of the multisubunit APC/C and two of the Class I suppressors are C. elegans homolog of APC/C activator CDC2O (FuRuTA et at. 2000; KITAGAwA  et at. 2002; TARAIL0 et al. 2007). In addition, previous works from multiple labs  have shown that reduced function of the majority of the APC/C components results in delayed meiotic and mitotic divisions (GoLDEN et a!. 2000; FURuTA et at. 2000; DAvis et a!. 2002; KITAGAwA  et al. 2002; RAPPLEYE et at. 2002; SHAKEs et at. 2003; TARAIL0 et at. 2007). Since,  none of the such genes are known SAC effectors, they are likely to identify genes not previously known to function in metaphase-to-anaphase transition in C. elegans. Alternatively, it is also possible that mutational inactivation of processes other than the metaphase-to-anaphase transition per se, for example, transcriptional regulation, could result in delayed mitosis. In human cancer cells, lesions in the retinoblastoma pathway lead to Mad2 overexpression through the E2F family of transcription factors. The aberrantly expressed Mad2 displayed a significant, two-fold longer mitosis, as a result of delayed degradation of securin and cyclinB (HERNAND0 et at. 2004). The suppressor analysis described in this thesis has identified a new APC/C component that rescues the mdJ4 (gk2) lethality. such-i encodes an APC5-like product not previously described. Many of the APC/C subunits have been identified in C. etegans (reviewed in YE0NG  81 2004; D0NG at a!. 2007). Previous work on the APC/C subunits led to proposal that the C. elegans gJI-3/APC5 and apc-]O/APC]O components do not function as a part of the meiotic APC/C (DAVIS at a!. 2002). The work in this thesis revealed that the C. elegans genome has two functional copies of APC5; gfi-3/M 163.4, which was previously described (ZAcHARIAE at al. 1998; DAVIS at a!. 2002) and such-i / Y66D 1 2A. 17 described here. The latter suppresses mdf 1 (gk2) lethality and delays anaphase onset, which clearly demonstrates its role in the metaphase to-anaphase transition. In addition, the data presented here indicate that simultaneous depletion of both copies of the APC5-like genes results in embryonic lethality at the meiotic one-cell stage. Furthermore, sequence analysis of the apc-1 0/Fl 5H10.3 gene revealed that this gene may have a paralog in C. elegnas as well. Although the functional redundancy of the apc-10/F15H1O.3 and the Y48G1C.12 genes was not confirmed, it is tempting to speculate that functional redundancy of the gfi-3 and ape-JO paralogs allows embryos to progress through meiosis and survive indefinitely. It is likely that this phenomenon obscured previous analyses and led to proposal that homologs of APC5 and APCJO genes may not be required for meiotic progression (DAVIS at a!. 2002). Furthermore, the sequence analyses revealed that gfl-3 and ape-JO are the only known APC/C components in C. elegans that have paralogs. It would be interesting to analyze the possibility of specialized roles of the paralogs. This type of analysis will become possible with the availability of mutant strains in all of these four genes. Unlike the Class I suppressors, the Class II suppressors, such-5, -6, and -7, are not likely candidates for the new APC/C components. The phenotypic analysis, mapping and sequencing data further support the hypothesis that these suppressors rescue the mdf-1 (gk2) lethality by an alternate mechanism. In contrast to the Class I suppressors, the such-5 and such-6 suppressors do not display Him phenotype in a wild-type background. Furthermore, the such-6 is the only suppressor that exhibits developmental delay phenotype. In addition, the such-7 is the only suppressor that is defective in DDR response, more precisely it abrogates the DNA damage-  82 induced apoptosis. Although, it was shown that it is unlikely that loss of apoptosis alone could rescue the mdJ] (gk2) lethality (Chapter 2 this thesis), the possible explanation that could account for this class of suppressors is that mutations in genes involved in checkpoint activity could partialy compensate for the MDF-l loss. For instance, it is possible that such-7 mutant abrogates both the cycle arrest and apoptosis induced by DNA damage. Previous studies have shown that the absence of DNA damage sensors, such as HUS-1, MRT-2 and RAD-5, results in a failure to arrest cell cycle and inability to promote apoptosis induced by DNA damage (reviewed in STERGI0u and HENGARmER 2004). Thus, it is possible that by abolishing the cell cycle arrest such-7 allows the germ cells to proliferate even in the presence of DNA damage. Alternatively, it is possible that suppressors that bypass the MDF- 1 checkpoint requirement without delaying anaphase onset are unknown components of the mitotic checkpoint that partially restore the SAC activity in the absence of MDF- 1 to delay progression through mitosis in the presence of chemical or mutational disruptions of the mitotic spindle. On the other hand, mutations in the components downstream of APC/C could rescue mdf I lethality without securin accumulation; however, mutants in the downstream components would be expected to delay anaphase onset. In conclusion, phenotypic analysis, mapping and sequencing data further support the finding that suppressors fall into two classes. The Class I includes suppressor alleles of the known SAC effectors, emb-30/APC4 and fzy-1/CDC2O, newly identified such-1/APC5-like gene and potentially three new SAC effectors. The Class II suppressors will likely identify new genes important for genome integrity and potentially a novel mechanism of rescue of the mdf-1(gk2) lethality. In addition, the positional cloning and sequencing analysis suggests that the screen for suppressors of the mdf-1 lethal phenotype is not saturated. First, STEIN et a?. 2007 showed that mdfl(avl9) can rescue the lethality of mat-i, mat-2, mat-3, emb-27 emb-30 and emb-1. Only emb-30 was isolated in our screen. Second, from the eleven suppressors isolated in the screens,  83 there are at least nine different genes and the known suppressor genes emb-30 and fzy-1 were re isolated only twice. Furthermore, the majority of the suppressors map to regions where no known SAC pathway components have been identified. This suggests that the substantial number of genes can be mutated to bypass the spindle assembly checkpoint requirement.  84  3.5  BIBLIOGRAPHY  BRENNER,  S., 1974 The genetics of Caenorhabditis elegans. Genetics 77: 7 1-94.  CAssADA,  R., E. ISNENGHI, M. CuLL0TI and G. VON EHRENsTEII’4, 1981 Genetic analysis of  temperature-sensitive embryogenesis mutants in  Caenorhabdltis elegans. Dev. Biol. 1:  193-205. DAVIs,  E. S., L. WILLE, A. B. CHEsmuT, P. L. SADLER, D. C. SHAKES et al., 2002 Multiple subunits of the Caenorhabditis elegans anaphase-promoting complex are required for chromosome segregation during meiosis I. Genetics 160: 805-813.  D0NG,  Y., A. B0GDAN0vA, B. HABERMANN, W. ZAcI-IARIAE, and J. AHRINGER, 2007 Identification of the C. elegans anaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast. BMC Dev. Biol. doi: 10.1186/1471213X-7-19.  FuRuTA,  T., S. TucK, J. KIRcHNER, B. KocH, R. AuTY, Ret al., 2000 EMB-30: an APC-4  homologue required for metaphase to anaphase transition during meiosis and mitosis in Caenorhabditis elegans. Mol. Biol. Cell. 11: 1401-1419. GARCIA-MUSE,  T., and S. J. B0uLT0N, 2005 Distinct modes of ATR activation after replication  stress and DNA double-strand breaks in Caenorhabditis elegans. EMBO 24: 4345-4355. GoLDEN,  A., P. L. SADLER, M. R. WALLENFANG, J. M. SCHUMACHER, D. R. HAMILL et a!., 2000  Metaphase to Anaphase (mat) Transition-defective Mutants in Caenorhabditis elegans. J. Cell Biol. 151: 1469-1482. HERNANDO,  E., Z. NAHLE, G. JUAN, E. DIAz-R0DRIGUEz, M. ALAMIN0S et a!., 2004 Rb  inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature 430: 797-802. KITAciAwA,  R., and A. M. RosE, 1999 Components of the spindle-assembly checkpoint are  essential in Caenorhabditis elegans. Nat. Cell Biol. 1: 514-521.  85 KITAGAwA,  R., E. LAw, L. TANG and A. M. RosE, 2002 The Cdc2O homolog, FZY-l, and its  interacting protein, IFY- 1, are required for proper chromosome segregation in Caenorhabditis elegans. Cuff. Biol. 12: 2118-2123. L’HERNAuLT,  S. W., 2006 Spermatogenesis. WormBook, ed. The C. elegans Research  Community, WormBook, doi!1 0.1 895/wormbook. 1.7.1, http://www.wormbook.org. MCCARTER,  J., B.  BARTLETT,  T. DANG and T. SHEDL, 1999 On the control of oocyte meiotic  maturation and ovulation in Caenorhabditis elegans. Dev. Biol. 205: 111-128. PETERS,  J. M., 2006 The anaphase promoting complex/cyclosome: a machine designed to  destroy. Nat. Rev. Mo!. Cell Biol. 7: 644-656. RAPPLEYE  C.A., A. TAGAwA, R. LYczAK, B.  B0wERMAN  and R. V. AR0IAN, 2002 The  anaphase-promoting complex and separin are required for embryonic anterior-posterior axis formation. Dev Cell. 2:195-206. SHAKES,  D. C., P. L. SADLER, J. M. SCHuMAcHER, M. ABD0LRAsuLNIA and A. GOLDEN, 2003  Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities. Development 130: 1605-1620. STEIN,  K. K., E. S. DAVIS, T. HAYS and A. GOLDEN, 2007 Components of the Spindle Assembly Checkpoint Regulate the Anaphase-Promoting Complex During Meiosis in Caenorhabditis elegans. Genetics 175: 107-123.  STERGI0u,  L., and M. 0. HENGARTNER, 2004 Death and more: DNA damage response pathways  in the nematode C. elegans. Nat. Cell Death and Diff. 11: 2 1-28. TARAILO,  M., R. KITAGAwA and A. M. RoSE, 2007 Suppressors of spindle checkpoint defect  (such) mutants identify new mdl-] /MAD] interactors in Caenorhabditis elegans. Genetics 175: 1665— 1679.  86 VAN HAAFTEN,  G., R. R0MEJJN, J. POTHOF, W. K00LE, L. H. MuLLENDERs LH et al., 2006  Identification of conserved pathways of DNA-damage response and radiation protection by genome-wide RNAi. Cuff Biol. 16: 1344-13 50. VIsINTn.1,  R., S. PRINz, and A. AM0N, 1997 CDC2O and CDH1: a family of substrate-specific  activators of APC-dependent proteolysis. Science 278: 460-463. S. R., R. T. YEH, W. R. GIsH, R. H. WATERs0N and R. H. A. PLAsTERK, 2001 Rapid gene  WicKs,  mapping in Caenorhabditis elegans using a high density polymorphism map. Nat. Gen. 28: 160-164. YE0NG,  F. M, 2004 Anaphase-Promoting Complex in Caenorhabditis elegans. Mol. Cell Biol.  24: 2215-2225. ZAcHARIAE,  W., A. SHEvcHENK0, P. D. ANDREw5, R. CI0SK, M. GAL0vA, M. J. STARK, M.  MANN  and K. NA5MYTH, 1998 Mass spectrometric analysis of the anaphase-promoting  complex from yeast: identification of a subunit related to cullins. Science 279: 12161219.  87  Chapter 4’: Enhancers of the mdf-1(gk2) Lethal Phenotype 4.1  INTRODUCTION  The spindle assembly checkpoint is believed to respond to either a lack of tension or a lack of microtubule occupancy at kinetochores (WATERS et cii. 1998; STERN and MuRRAY 2001; LEw and BIJRKE 2003; HowELL et cii. 2004). The kinetochore plays an essential role in the SAC function. For example, mutations in centromeric DNA sequences and kinetochore proteins result in SAC-dependent mitotic delays (SPENcER and HIETER 1992; WANG and BuRKE 1995; PANQILmiAN  and SPENcER 1996; WELLS and MURRAY 1996). Furthermore, lesions in  kinetochore components display synthetic lethal phenotypes with checkpoint components in budding yeast (WANG and BURKE 1995; LEE and SPENCER 2004; DANIEL et cii. 2006). These and subsequent studies suggest that the kinetochore may sense damage and generate signals that activate the checkpoint to inhibit the Cdc2O-mediated activation of the APC/C. However, much is still unknown about the SAC pathway. For instance, the sensors of defective kinetochore microtubule attachment remain largely unknown. One way to better understand the SAC function is to define functional relationships between proteins by creating genetic interaction maps. We reasoned that upstream components required for the genome stability would be recognized as enhancers of the mdf-1 (gk2) lethal phenotype. In yeast, array analysis of deletion strains has been used to identif’ synthetic lethal interactions with the components of the SAC pathway (LEE and SPENcER 2004; DANIEL et a!. 2006). However, a similar approach is currently not feasible in a living animal. Unlike a  A version of this chapter has been accepted for publication:  M., S. TARAIL0 and A. M. ROSE, 2007 Synthetic Lethal Interactions Identify Phenotypic “Interologs” of the Spindle Assembly Checkpoint Components. Genetics 177: 2525 2530.  TARAIL0,  -  88 suppressor screen, a genetic screen for mutations that enhance the mdfil (gk2) lethality would be difficult. First, mdf-1 (gk2) mutation results in lethality after the second generation. Second, many genes when mutated result in lethality even in the absence of the checkpoint deletion, producing a large background of apparent enhancers, emphasizing the impracticality of genetic screens. Furthermore, a screen would have to be designed to maintain the enhancer mutations for subsequent cloning, greatly adding to the labour involved. In this thesis, enhancement of mdf-1(gk2) lethality is described. To avoid the difficulties associated with a genetic screen for mutations that enhance mdJ4(gk2) lethality, RNAi by the feeding method (TIMM0Ns and FIRE, 1998) is used to screen for gene disruptions which are synthetic lethal with mdf-1(gk2).  89  4.2  4.2.1  MATERIALS AND METHODS  C. elegans strains, alleles and culturing The Bristol strain N2 was used as the standard wild-type strain (BRENNER 1974). The  marker mutations and balancer chromosomes used are listed in the order of chromosomes. LGI: dpy-5(e61), dpy-5(s]300), LGII: dpy-1O(e128); LGIII: dpy-] 7(e]64); LGIV: dpy-13(e184) and LGV: unc-46(el 77), mdf-1(gk2) and nT](IJ’ V). The strains used include: KR3627 unc-46(el 77) mdf-](gk2) +/+ + nTl [let-X]; KR379 dpy-5(e6]) rec-](s180); KR4303 unc-46(e]77) mdf ](gk2) IV /nT][let-?(m435)] (IV V), unc-]]9(ed3) ruIs32[unc-119(+) pie-1::GFP::H2BJ III; KR4144 unc-46(e]77) +/+ + nTl [let-X]; CB1511 him-1O(el5llts)III and RB1391 san ](ok]580) I. The RB1391 strain was obtained from the Caenorhabditis Genetics Center. The mdf-2(tm2910) allele was kindly provided by the Bioresource Project (Shohei Mitani). The mdf 2(vcl5) allele was generated in our laboratory using tilling method (GILcHRIsT et a!. 2007). Animals were maintained using standard procedures (BRENNER 1974). 4.2.2  Measuring the level of enhancement of mdf-1(gk2) lethality 1 generation, L4 wild-type worms of genotype For analysis of the enhancement in F  (unc-46(e] 77) mdf-1(gk2) +/+ + nTl [let-X]) were individually plated. The worms were transferred to fresh plates every 12 hours and the progeny that developed to adult stage were scored to determine the ratio of wild-type to unc-46 mdJl worms. The Unc-46 Mdf-1 to WT 1 is recognized as lower than 25% of ratio is 1:4 in KR3627 strain. The enhancement in the F Unc-46Mdf-1 animals. The observed over expected (25%) ratio was then calculated. For analysis 2 generation, 15 L4 unc-46 mdf-1 homozygous worms were plated. The of the enhancement in F worms were transferred to fresh plates every 12 hours and the plates were scored. The eggs that did not hatch in 24 hours were scored as embryonic arrest. The eggs that hatched but did not reach adult stage were scored as larval arrest.  90 4.2.3  Detecting putative orthologs To identify putative orthologs of yeast proteins in C. elegans, reciprocal best  BLASTP  (ALTscHUL et a!. 1997) hit (RBH) approach was used. In summary, with the RBH method, protein x in genome X is considered a putative ortholog of protein y in genome Y if BLAST P of protein x against genome Y yields as the top hit protein y, and reciprocal  BLAST P  of protein y  against genome X yields as the top hit protein x. In this analysis, in case of alternative isoforms for a gene, only the longest peptide isoform was considered, so that one-to-one gene to peptide relation is maintained. 4.2.4  RNA interference assay As described in Chapters 2 and 3 MATERIALS and METHODS  All the RNAi bacterial strains for genes not located on chromosome I were obtained from either Baillie or Riddle Labs. 4.2.5  Measuring viability in mdf-2, san-i and him-i 0 mutants The mdf-2(tm2i9O), san-i (ok1580) and him-iO(ei5iits) animals were fed dsRNA.  Their progeny were plated individually in L4 stage on the RNAi plates and allowed to lay eggs for 12 hours. The adults were then removed and the embryos were analyzed for the ability to reach adult stage. Incidence of males was measured as number of males observed divided by the total number of adult progeny. 4.2.6  Suppression assay All of the suppressor strains in the mdfi] (gk2) background, as described in Chapters 2  and 3, were grown (10 worms per plate) on the hcp-i, hcp-2, bub-3 and control RNAi plates. The worms (10 from each plate) were transferred every generation for four generations to fresh plates and analyzed. All the plates that survived to forth generation were scored as positive for suppressors of the enhanced mdf-i(gk2) lethality.  91  4.3  4.3.1  RESULTS  Enhanced mdf-1(gk2) lethality Previously, it was reported that mdf-](gk2) homozygotes, segregated from a  heterozygous parent, had no obvious phenotype in the first generation, most likely due to maternally supplied MDF-1 protein (KITAGAwA and RosE 1999; TARAIL0 et a!. 2007a). However, in subsequent generations genetic errors arise and accumulate leading to genetic lethality (KITAGAwA and ROSE 1999; TARAIL0 et al. 2007a). The number of defects observed increased with increasing numbers of cell divisions and the strain could not be maintained past third generation (KITAGAwA and ROSE 1999). The pleiotropic range of phenotypes observed in the absence of MDF- 1 was observed in several different genetic backgrounds, suggesting that the primary phenotype is chromosome instability (CIN) (KITAGAwA and RosE 1999; TARAIL0 et a!. 2007a). This was the first demonstration of the effects of a defective checkpoint in whole animal development (KITAGAwA and RosE 1999). 4.3.1.1  2 generation Enhanced mdf-1(gk2) lethality in F  How do we recognize enhanced mdf-1(gk2) lethality? An answer to this question came from a temperature study, where it was shown that higher temperature (25°C) intensified the effect of the mdf-1(gk2) (TARAILO et al. 2007a; Table 2.1). The mdf-1(gk2) homozygotes cannot be maintained beyond the first generation at 25°C (TARAIL0 et a!. 2007a). Furthermore, during the course of mapping the suppressor mutations to a chromosome, lower survivability of the suppressor strains was observed in the presence of dpy-1O and dpy-5 phenotypic markers. To test whether the phenotypic markers have an effect on the mdJl(gk2) mutant, all of the markers were crossed into the KR3627 strain and DpyUnc-46Mdf-1 progeny were plated and scored for phenotypes (Figure 4.1). Analysis of these mutants revealed that,  92  25  CA)  -  20  4.  D  15  -  >%  10  -  CD  >  50  -i-—  Figure 4.1: dpy-1O(e128) and dpy-5(e51) enhance the mdf-1(gk2) lethality. At 20°C the mdf-1(gk2)  homozygotes cannot be maintained past F 3 and in the dpy-1O(e128) and dpy-5(e51) background at 20°C mdf-1(gk2) homozygotes cannot be maintained past F , since only a small number of F 2 2 progeny reach adulthood.  while the dpy-] 7(e] 64) and dpy-]3(e]84) had no obvious effect on mdf-] (gk2) lethality, the dpy ]O(e128) and dpy-5(e51) mutants resulted in significant enhancement of the mdfil(gk2) lethal phenotype (Figure 4.1). Recently, STEIN et a!. (2007) reported that dpy-]O(e]28) was able to suppress the lethality of the mat-3(orl8O)/APC8 and attributed this interaction to induction of physiological stress response. Since the KR379 strain of dpy-5(e61) was recently found to contain the rec-] (s180) mutation, another dpy-5 mutant was tested for its ability to enhance the mdfl(gk2) lethality. Interestingly, dpy-5(s1300) mutant had no apparent effect on the mdf.1(gk2) lethality (Figure 4.1), suggesting the possibility that another mutation present in the KR379 strain may be responsible for the observed enhancement. This mutation was given a new allele  93 name, h2150, and tested for linkage to chromosome I. Fifty Dpy-5Unc-46Mdf-l animals were tested and one non-lethal individual, a potential h2150 recombinant was observed. The data are consistent with linkage to dpy-5 on chromosome I. 4.3.1.2  Enhanced mdf-1(gk2) lethality in F 1 generation  In order to study chromosome dynamics in mdfl(gk2) homozygotes, the KR4303 strain of genotype unc-46(e] 77) mdf-](gk2) V /nT][let-?(m435)] (IV; V), unc-119(ed3) ruIs32[unc119(+) pie-]::GFP::H2B] III was constructed  (TARAIL0  et al. 2007a). Analysis of this strain  revealed a decrease in unc-46(e] 77) mdJ4(gk2) F 1 homozygotes, when compared to the KR3627 strain (unc-46(el 77) mdf-1(gk2) V/nTl[let-?(m435)] (JV V)) that does not contain the transgene (Figure 4.2). The KR3627 strain segregates mainly developmentally arrested progeny (inviable nTl), wild-type progeny with the parental genotype unc-46(el 77) mdfi](gk2) homozygotes. The Unc-46Mdf-1 to WT ratio is 1:4 in KR3 627 strain. However, this ratio is altered in the KR4303 strain (1:10), suggesting lethality of the mdfil(gk2) homozygotes in F 1 generation (Figure 4.2). The toxic effect of the transgene is specific to mdJl (gk2) homozygotes, since the transgene has no effect on the unc-46(e177) mutation (Figure 4.2). Thus, it is likely that the H2B::GFP transgene affects chromatin structure in a way that it increases mis-segregation. In the absence of the SAC checkpoint to compensate for the mis-segregation defect, first generation lethality is observed. This dramatic phenotype creates a powerful assay that can be used to screen for  enhancers of mdf-1(gk2) lethality. 4.3.2  Identification of conserved mdf-1/MAD1 genetic interactions Due to the variability of the mdfl(gk2) phenotype in the second generation, the screen  to identify nonessential genes that, when disrupted, enhance mdJl(gk2) lethality was performed by scoring the enhanced mdJ](gk2) lethality as decreased F 1 viability. The known MAD] synthetic lethal interactions observed in yeast were examined for their ability to enhance mdf.1(gk2) lethality in C. elegans  (TARAIL0  et al. 2007b). To date, there  94  100 90  E .  0  ax  w  80 70 60 50 40 30  .0  o  20  110 0 a  -  0  C;  Figure 4.2: An integrated H2B::GFP transgene, ruIs32, enhances the mdf-1(gk2) lethality in F . 1  Observed over expected ratio of the Unc-46 worms to WT-looking worms. We analyzed worms of three different genotypes: KR3627 strain (unc-46(e177) mdf-1(gk2) V /nTl[let-?(m435)] (IV;V)), KR4303 strain (unc-46(el 77) mdf-] (gk2) V /nTl [let-? (m435)] (IV; V); unc-] 19(ed3) ruIs32[unc-] ]9(+) pie 1::GFP..JI2B] II]) and control strain (unc-46(e 177) V /nTl[let-?(m435)] (IV;V); unc-119(ed3) ruIs32[unc-119(+) pie-1::GFP::H2B] III by individually plating ten L4 wild-type looking worms from each strain. The progeny that developed to adult stage were scored for the ratio of Unc-46 to WT-looking worms, this ratio was then divided by the 1:4 ratio expected to be observed. All the measurements were performed at 20°C.  are 79 MAD] synthetic lethal interactions identified in S. cerevisiae (WANG and BuRKE 1995; HARDwIcK et al. 1999; ToNG et al. 2004; LEE and SPENcER 2004; KR0GAN et al. 2004; MEASDAY et al. 2005; M0NTPETIT et a!. 2005; BLAKE et al. 2006; DANIEL et al. 2006; PAN et a!. 2006). The reciprocal best  BLASTP (ALTscHuL  et al. 1997) hit (RBH) approach was used to  identify putative C. elegans orthologs for 37 of these 79 genes (TARAIL0 et a!. 200Th). Of the 42  95 genes, 14 had matches to multiple C. elegans proteins, while 28 did not display significant homology to any of the proteins in the C. elegans genome. Of the 37 putative orthologs, seven were not present in the C. elegans RNAi library (KAMATH et al. 2003) and nine, ssl-i, C3OC1 1.4, klp-3, prp-i 7, pyp-], pfd-3, pfd-5, pfd-6 and F56B3.8 were lethal when inactivated by RNAi, in both experimental and control strains and therefore could not be assessed for enhancement (TARAIL0 et a!. Yl 1 1B2A.8/SNF4, Ri 3F6. 1O/MDM2O,  2007b).  CO8B1 1 .6/ARP6, W08F4.3/ERG2,  Of 21  putative orthologs tested,  spt-4/SPT4, pfd-4/GIM3,  C34G6.5/CDC7, K08F4. 1 /CTF1 8,  12 genes: cdk-8/SSN3,  F34D 1 0.2/CDC45,  F59A3.2/CSE2 and cdk-5/PH085 did not display enhanced mdf-i(gk2) lethality (TARAIL0 et at. 2007b). The remaining nine of the 21 non-lethal candidates were strong enhancers, resulting in a significant alteration of Unc-46 to WT ratio in the KR3627 strain, but not in the control KR4144 strain (Figure 4.3). Thus, nine of the synthetic lethal interactions observed in yeast are conserved in C. elegans. These conserved genetic interactions will be referred to as “genetic interologs”, a term adopted from WALH0uT et al. (2000) who used interolog to describe physical interactions conserved between species (TARAIL0 et at. 2007b).  4.3.3  Enhancement of the other checkpoint mutants: To investigate whether the confirmed genetic interologs display specificity for the mdf  1 (gk2)/M4D1 component of the spindle checkpoint or a more general effect on the SAC pathway, the enhancer phenotypes were tested in the absence of SAC components. To date, knockout alleles have been isolated in four components of the spindle checkpoint: mdJi, mdJ2, san-i and bub-i (KITAGAwA and RosE 1999; STEIN et a!. 2007). The enhancers were tested for interactions with mdJ2(tm29iO)/MAD2 and san-i (ok1580)/MAD3 knockout alleles; the bub i(tm28]5)/BUBJ deletion allele results in lethality and could not be tested.  96  E a) 0  a)  2.  >< LU  a)  > a.) .2  0  a) 0  a)  Figure 4.3: Enhancers identify conserved mdf-1/MAD1 genetic interactions. The figure represents a  plot of observed over expected ratio of the Unc-46 Mdf-1 worms to WT-looking worms for all of the nine enhancer genes. All the RNAi vectors were tested by PCR amplification. In the cases where altered Unc 46 to WT-looking worms ratio was observed in the KR3627 strain, but not in the KR4144 strain, the RNAi clone was scored as positive for enhancement. The data obtained is from four independent experiments with more than 2000 animals assayed. This work was carried in part by a Research Assistant Sanja Tarailo. For all experiments animals were kept at 20°C.  To address the effect of deletion of the md2 checkpoint gene, the mdJ2 deletion allele tm29]O was tested. The tm29]O deletion removes 864 nucleotides between intron 3 and exon 6  and is likely to be a null mutation. In contrast to mdf-] (gk2), mdfi2(tm291 0) homozygotes are not ultimately lethal and can be maintained at 20°C indefinitely. They do however display severely  97 reduced brood size (33 progeny) of which 19% arrest as embryos, 36% arrest as larvae and 45% develop into adults (Table 4.1 and Figure 4.4). The strain also has a high incidence of males (Him) phenotype (3% of the adult progeny are males), which is an indicator of chromosome missegregation. Although the absence of MDF-2 has a profound effect on C. elegans development and a clear effect on genome stability, MDF-2 is not essential for C. elegans survival under normal conditions (TARAIL0 et al. 2007b). Similarly, san-i is not essential for C. elegans survival as the san-i (okiS8O) deletion allele has only a mild phenotype resulting in only -43% lethality (STEIN et al. 2007; Table 4.1 and Figure 4.2). Analysis of the nine mdf] (gk2) enhancers revealed that six of these significantly enhanced the lethality of mdJ2(tm29i 0) (Table 4.1). All six enhancers are synthetic lethal interactions with MAD2 in yeast and thus represent functionally conserved genetic interologs (Figure 4.5). Furthermore, three of the six mdfi(gk2) and mdfi2(tm29iO) interactors enhanced the lethality of san-i (oki 580) and represent conserved genetic interactions of MAD3 (Table 4.1; Figure 4.5). None of the 12 genes that failed to enhance the mdf 1 (gk2) lethality enhanced the lethality of either mdf-2(tm29iO), or san-i (ok1580) (data not shown). Further evidence that the enhancers have a specific effect on the checkpoint came from the analysis of their general effect on kinetochore. None of the analyzed genes enhanced the lethality of a mutant in the kinetochore component, him-iO(e15] lts)/NUF2 (Table 4.1). Thus, the identified enhancers have a specific effect on the spindle checkpoint (TARAIL0 et al. 2007b).  4.3.4  Y54G9A.6/BUB3 enhances the lethality of SAC mutants A putative ortholog of the kinetochore-associated component Bub3, Y54G9A.6 was  tested (reviewed in OEGEMA and HYMAN 2006; TARAIL0 et a!. 2007a; STEIN et a!. 2007). RNAi for the gene, named bub-3 (TARAIL0 et a?. 2007b) produced no obvious phenotypes (Figure 4.4B); however, depletion of BUB-3 in the absence of either MDF-l, MDF-2 or SAN-l results in  98  TABLE 4.1 Genetic interaction between the mdf-1/MAD1 interologs and the mdf-2/M4D2 and san 1/MAD3 checkpoint components dsRNA  mdJ2(tm2190)  san-i (ok1580)  him-lO(ei5lits)  Percentage of adults ± SE  Percentage of adults ± SE  Percentage of adults ± SE  Control RNAi  45.0±2.8  86.8±5.0  63.8±8.8  F39H11.l/SWC5  39.4±8.2  71.0±15.4  69.4±5.6  Jkh-iO/HCMI  24.5±8.0*  91.5±5.7  73.8±1.9  F23C8.9/CSM3  49.0±6.2  73.6±9.0  64.6±4.0  K07H8. I /PAC2  18.6±7.3*  54.8±9.3*  75.0±4.0  pfd-2/GIM4  3.5±2.3*  19.2±14.3*  54.6±12.5  F16D3.4/CIN1  0.2±0.2*  0.2±0.2*  59.2±3.5  K09H9.2/DCCJ  30.3±6.6*  91.4±6.2  55.8±5.4  Y39B6A.39/RSM24  22.3±8.2*  97.8±5.6  67.8±2.3  F3 I C3 .2/PAP2  39.4±8.2  79.4±4.2  65.0±3.2  For each mutant strain and dsRNA, the data obtained represent three independent experiments with more than 1000 embryos analyzed. The bold numbers and asterisks denote a significant difference between the experimental animals and the control animals. This work was carried in part by a Research Assistant Sanja Tarailo. All the experiments were performed at 20°C.  a significant decrease in viability, presumably due to elevated chromosome instability (Figure 4.4). Loss of both BUB-3 and SAN-i was observed to cause an increase in chromosome missegregation; san-i (ok1580) mutants produced 1% males and the incidence of males was increased twofold in the san-i(ok1580); bub-3(RNAi) animals (TARAIL0 et al. 2007b). In yeast,  BUB3 has not been shown to be synthetic lethal with any of the SAC mutants (Figure 4.5). Thus, this is the first description of its interaction with the checkpoint (Figure 4.5). These data provide  99 the first evidence for the function of this gene in C. elegans, BUB-3 functions within the SAC pathway to ensure proper cell cycle progression (TARAIL0 et al. 2007b).  4.3.5  hcp-1/CENP-F enhances the lethality of SAC mutants We asked whether kinetochore defects would enhance lethality in the absence of MDF  1. In C. elegans, most kinetochore components identified to date are essential (reviewed in OEGEMA and HYMAN 2006). The exceptions are kbp-5, hcp-] and hcp-2. Analysis of these genes revealed that, depletion of either KBP-5 or HCP-2 had no obvious effect on mdf-1(gk2) lethality; however, depletion of HCP-1 resulted in significant enhancement of the mdfi(gk2) lethal phenotype (Figure 4.4A). In human cells, CENP-F (mitosin), a large 35OkDa transient kinetochore component, associates with the outer kinetochore (RArrNR et al. 1993; LIA0 et al. 1995; ZHu et a?. 1995). Although the precise function of CENP-F is unknown, an emerging body of evidence implicates CENP-F in kinetochore maturation, regulation of chromosome behavior, and control of SAC  activity (EAKER et a!. 2001; LA0uKILI et al. 2005; H0LT et a?. 2005; YANG et a?. 2005). In C. elegans, HCP-1 and HCP-2, two CENP-F-like proteins, were shown to contribute redundantly to the fidelity of chromosome segregation (MOORE et a?. 1999) and to the SAC response in the presence of either chemical or mutational disruptions of the microtubule cytoskeleton (STEAR and RoTH 2004; ENcALADA et a?. 2005). CHEEsEMAN et a?. (2005) proposed that HCP-1 and HCP-2 function redundantly to target the microtubule-associated protein CLASP/CLS-2 to kinetochores, where it may function to promote the polymerization of kinetochore bound microtubules. Depletion of either HCP-1 or HCP-2 alone does not result in significant developmental arrest in a wild-type background, while co-depletion of both proteins results in highly penetrant embryonic lethality caused by chromosome segregation defects, inability to  form metaphase plates and precocious anaphase  100 A  B 100 0  .8  0 0 0  90 80 70 50  Lii 0 0  40  S  S  0 .0  30  0  20  0  0.  0 0  c_  C  D 50 45 40  100  35 0  30  !.  25 20 15 10  1  —  —  I  1 80 70 50 40 .9 30 .0  >20  C.’ ’ 0 Cl  9 ,ç.  Figure 4.4: hcp-1(RNAi) and bub-3(RJVAi) enhance the mdf-1(gk2) lethality. (A) Observed over  expected ratio, Unc-46 Mdf-l worms to WT-looking worms, in the KR3627 strain treated with cRNA (vector with no insert), kbp-5(RNAI), bub-3(R]’JAz), hcp-i(R1’/Ai) and hcp-2(RNAi). The data obtained is from at least four independent experiments with more than 2000 animals assayed. (B) The wild-type animals were fed cRNAi, bub-3(RNAz), hcp-I(RNAi), hcp-2(RNAz) and hcp-i/2(RA Ai) and the (C) mdf T 2(tm2910) and (D) san-i (ok1580) mutants were fed cRNAi, bub-3(R1’/Ai), hcpi(RA1Ai and hcp-2(RATAz).  For each dsRNA and mutant strain, the data obtained represent three independent experiments with more than 1000 embryos analyzed. Bars represent the mean with SE error bars. This work was carried in part by a Research Assistant Sanja Tarailo. The experiments were performed at 20°C.  onset (MOORE et al. 1999; ENcALADA et al. 2005; Figure 4.4B). I show here that depletion of HCP- 1 in the absence of either MDF- 1, MDF-2 or SAN-i resulted in a significant decrease in viability (Figure 4.4). Unexpectedly, depletion of HCP-2 had no obvious effect on viability of the mdf-i(gk2), mdf-2(tm2910), or san-i (ok1580) mutants (Figure 4.4). A mutant strain of mdf  2(vciS) generated by GILcHRIsT et al. (2007) was used to analyze the fidelity of chromosome segregation in a SAC mutant in the absence of HCP-i. The mdf-2(vci5) homozygote has a  101 K07H8.1  fkh-1O  W08F4.3 F34D1 0.2 13F6.1O  Y39B6A.39  Figure 4.5: Summary of the genetic interaction data. Arrows and circles/squares represent synthetic  lethal interactions and C. elegans genes, respectively. Circles represent genes of known function, while squares represent genes of unknown function in C. elegans. The color-code for interactions is as follows: red arrows represent detected interologs; black arrows represent synthetic lethal interactions in yeast that were not observed in C. elegans and blue arrows represent novel interactions identified in worm and not observed in yeast. ORTIZ et at (1999) suggested homology between Okpl and CENP-F. EVANS et al. (2007) suggested homology between Okpl and hcp-1. Okpl does not display synthetic lethality with any of the SAC components in yeast.  milder phenotype than the mdf-2(tm2910) animals, as 89% of progeny develop into adults, of which 0.2% are male making it easier to identify increased chromosome instability. While depletion of HCP-2 had no effect on mdf-2(vclS) worms, depletion of HCP-1 resulted in a significant decrease in viability, as only 59% of progeny develop into adults, of which 2% are  102 male. This result suggests that synthetic lethality between hcp-1 and SAC components results from chromosome instability. Furthermore, HCP- 1 and HCP-2 do not contribute equally to the stability and do not have completely redundant functions (TARAIL0 et al. 2007b). 4.3.6  Ability of mdf-1 suppressors to rescue enhanced mdf-1 lethality In human cells, CENP-F functions downstream of BUB1 and upstream of MAD1 and  MAD2  (JoHNsoN  et a?. 2004;  LA0uKILI  et a?. 2005). A mitotic checkpoint complex (MCC) that  contains BUBR1, MAD2, CDC2O and BUB3, has been shown to be about 3000-fold more potent at inhibiting anaphase onset than MAD2 alone  (SuDAKIN  et al. 2001). In C. elegans, depletion of  either HCP-1 or BUB-3 by RNAi resulted in significant enhancement of mdf.] (gk2) lethality (Figure 4.4). To investigate whether any of the suppressors could reverse this result, viability in the absence of either HCP-1 or BUB-3 was investigated. Ten L4 hermaphrodites for 11 mdf ](gk2);suppressor lines, described in  TARAILO  et al. (2007a) and Chapters 2 and 3 of this thesis,  were fed hcp-] or bub-3 RNAi-expressing bacteria for four generations by transferring viable progeny from each generation to fresh RNAi plates. As a control, an RNAi construct with no insert and hcp-2 RNAi-expressing bacteria were used. The suppressor lines displayed normal mdf-1(gk2);suppressor viability on the control RNAi plates (Table 4.2). The majority of suppressors failed to reverse the mdf-] (gk2) hcp-] (RNAt) lethality (Table 4.2). However, three of the suppressor strains,fzy-](h]983), such-] (h]960ts) and such-6(h]958) remained viable for four generations on hcp-] RNAi. Thus in the absence of both kinetochore components, HCP-l and the spindle checkpoint component, MDF- 1, which results in arrested division, three interactors  are identified that can partially rescue the lethal phenotype. Apparently, these suppressors were able to bypass the requirement for wild-type HCP- 1 in animals that lack MDF- 1.  103 TABLE 4.2 Suppression of the synthetic lethality of HCP-1- or BUB-3-depleted mdf-1(gk2) animals Genotype  Control RNAi  hcp-1(RNAi)  hcp-2(RNAi)  bub-3(RNAI)  mdf-1(gk2);emb-30(h1959ts)  Sup  Let  Sup  Sup  mdf-I(gk2),emb-30(h1962ts)  Sup  Let  Sup  Sup  mdf4(’gk2);such-l(hJ96Ots)  Sup  Sup  Sup  Sup  mdf1(gk2);such-6(h1958)  Sup  Sup  Sup  Sup  mdf-1(gk2),fzy-](h1988ts)  Sup  Let  Sup  Sup  mdf-1(gk2)such-2(h1992)  Sup  Let  Sup  Sup  mdf-1(gk2);such-5(h1987)  Sup  Let  Sup  Sup  mdf-1(gk2);fzy-1(h1983)  Sup  Sup  Sup  Sup  mdf-I(gk2)such-7(h1985)  Sup  Let  Sup  Sup  mdJ4(gk2);such-3(h1989)  Sup  Let  Sup  Sup  mdf-I(gk2) such-4(h2168)  Sup  Let  Sup  Let  mdf-1(gk2), suppressor L4 animals (10/plate) were fed control RJ’/Ai (vector with no insert), hcp  1 (RNAi), hcp-2(RNAz) or bub-3(R1’/Ai) and their progeny were followed for four generations for the ability to survive. All the experiments were performed at 20°C. Sup denotes suppressed; Let denotes lethal. Most suppressor strains failed to suppress mdf-1(gk2) lethality when HCP-l was depleted.  FZY- 1 regulates timing of anaphase onset and may rescue the observed lethality by delaying anaphase onset. Similarly, such-i (hi96Ots), along with fzy-1(hi983) results in the biggest delay in anaphase onset, when compared to all other suppressors in this group (Figures 2.1 and 2.2). On the other hand, such-6 (hi 958) does not display delay in anaphase onset, but is a strong suppressor of unknown function. These data are consistent with MDF-1, SUCH-6, SUCH 1 and FZY- 1, all being downstream of HCP- 1. Furthermore, most of the suppressors rescued mdJi(gk2); bub-3(RNAz) enhanced lethality for four generations except for the weakest Class I suppressor such-4(h2i 68) (Table 2.1  104 and Figure 2.2). The data are consistent with a role for BUB-3 downstream of HCP-1 and upstream of APC/C. In summary, the C. elegans data are consistent with those from human cells in that hcp-]/CENP-F functions upstream of MDF- 1 [MAD 1, while BUB-3/BUB3 functions as part of the checkpoint, ensuring proper checkpoint response.  105  4.4  DISCUSSION  In this thesis, non-essential genes that enhance the lethality of mdf 1 (gk2) have been identified. Using a mutant in the checkpoint component and RNAi for 21 non-essential putative orthologs nine genetic interactions with mdf-1/MAD1 were confirmed (Figure 4.3). Two previously unknown interactions, hcp-1 and bub-3, were characterized (Figure 4.3). Data presented in this thesis revealed the unexpected result that HCP-1 and HCP-2, two CENP-F related proteins, recently implicated in the spindle assembly checkpoint function, do not have identical functions. hcp-1(RNAz), but not hcp-2(RNAz), enhanced the lethality of checkpoint mutants. 21 non-essential putative orthologs were tested and 12 did not display enhanced mdf 1 (gk2) lethality. There are several possible explanations for the inability of these synthetic lethal MAD] interactions to enhance mdJl (gk2) lethality in C. elegans. First, it is possible that some of the putative orthologs identified by the sequence analysis are not functional orthologs of these genes in C. elegans. A common procedure and widely used method for identifying sequence pairs that are putative orthologs is the reciprocal best  BLAST P (ALTscHuL  et a!. 1997) hit (RBH)  method. The RBH method is stringent and succeeds in the selection of conserved orthologs with a low false positive rate, but it cannot predict the functional roles of paralogs (FuLT0N et a!. 2006). A second explanation for the lack of enhancement may be variability in the effectiveness of the RNAi treatment. In some cases the lower efficiency of RNAi for some genes may have produced a false negative result. A third possibility is that the function of the putative orthologs is differentiated in a multicellular organism and that loss of these genes does not result in organismal lethality, but rather has a consequence on a specific tissue. MATTHEws et a!. (2001) performed systematic BLAST analysis to identify pairs of putative orthologs of known proteinprotein interactions in S. cerevisiae to identify potentially conserved physical interactions in C.  106 elegans. These authors suggested that only 16% to 31% of physical interactions would be detected using this approach for these two species which are evolutionarily distant by 900 million years (MATrHEws et al. 2001). In this thesis of 21 putative interactions, nine scored positive, a 43% success rate. Thus, similar to protein interaction maps, the genetic interaction maps from one species are useful in predicting interactions in another species (TARAIL0 et al. 2007b). Checkpoint proteins are likely to be involved in functions other than anaphase onset monitoring. KITAGAwA and RosE (1999) observed MDF-1-independent MDF-2 localization in gut-cell precursors, suggesting a possible role of MDF-2 in endoreduplication. During anoxia induced suspended animation, embryos lacking functional SAN-1/Mad3 or MDF-2/Mad2 fail to arrest the cell cycle (NYSTUL et al. 2003). BUB-1/Bubl was shown to localize to kinetochores and to have an essential role in kinetochore function as well as a potential role in regulating chromatin cohesion (OEGEMA et a!. 2001; DEsAI et al. 2003; M0NEN et a!. 2005). Different phenotypic consequences of inactivation of SAC genes also support different roles of these genes in C. elegans. For instance, MDF-2 is not essential for C. elegans survival (TARAIL0 et al. 2007b) san-i (ok1580)/MAD3 and bub-3(RNAi)/BUB3 have mild non-essential effect on development and genome stability. On the other hand, depletion of BUB- 1 results in embryonic lethality, consistent with its essential role at the kinetochore independent of its checkpoint function (DEsAT et a!. 2003). MDF-1 is the only SAC component that when depleted displays lethality due to accumulation of mis-segregated chromosomes at each cell division (TARAIL0 et al. 2007b). MDF-1 is an essential component of the SAC and this may be its primary role. Six of the mdf 1/MAD] genetic interologs identified enhanced the mdf-2(tm291 O)/MAD2 lethality; and three also enhanced the san-i (oki58O) lethality (Figure 4.3). None enhanced lethality in the kinetochore mutant him-iO(ei5iits)/NUF2. A spectrum of genetic interactions of SAC genes  107 supports additional roles for these genes. Interaction data for mdf-i, mdf-2 and san-i support the findings that some synthetic interactions that are common to MAD] and MAD2 are not shared by MAD3 (LEE and SPENcER 2004; DANIEL et a!. 2006; Figure 4.3), indicating that, in C. elegans as in yeast, MDF-1/Madl and MDF-2/Mad2 have functions that are not shared by SAN-1/Mad3 and that MDF-1 and MDF-2, although closely related, differ in phenotype and the spectrum of genetic interactions, suggesting overlapping but not identical roles in C. elegans (TARAIL0 et al. 2007b). Two previously unknown interactions were identified in C. elegans and are not conserved in yeast (Figure 4.3). The Y54G9A.6 gene was identified as a putative ortholog of the kinetochore-associated SAC component Bub3 by sequence homology (TARAIL0 et al. 2007a; STEIN  et a!. 2007a). Data presented in this thesis provide the first evidence regarding the function  of this gene in C. elegans. Unlike in yeast, depletion of BUB-3 displays synthetic enhanced phenotype with mdf-](gk2), mdf-2(tm2190) and san-i (ok]580) (TARAIL0 et a!. 2007b). These results suggest that BUB-3 may provide some residual checkpoint activity in the absence of MDF- 1, MDF-2 or SAN-i. Alternatively, BUB-3 may function upstream of these SAC components and have a role in chromosome stability in addition to its function in SAC. Finding that in the absence of both SAN-i and BUB-3 chromosome instability is increased twofold would support both of these hypothesis. However, the ability of the suppressors to rescue mdf 1(gk2); bub-3(RNAi) enhanced lethality favors the first hypothesis, and supports a role for BUB 3 in the core checkpoint function. hcp-] has been identified as the strongest enhancer of mdf-1 (gk2), mdf-2(tm2]90) and san-i (ok]580) lethality (TARAIL0 et a!. 2007b). In human cells, CENP-F functions upstream of MAD1 (JoHNsoN et al. 2004; LA0UKILI et a!. 2005) and is important for kinetochore maturation, regulation of chromosome behavior, and control of SAC activity (EAKER et a!. 2001; LA0uKILI et al. 2005; H0LT et al. 2005; YANG et a!. 2005). In C. elegans, two CENP-F-like proteins, HCP  108 1 and HCP-2, were shown to contribute redundantly to the fidelity of chromosome segregation (MOORE et al. 1999) and to the SAC response in the presence of either chemical or mutational disruptions of the microtubule cytoskeleton (STEAR and ROTH 2004; ENcALADA et al. 2005). In yeast, Okpl was suggested as a homolog of CENP-F and hcp-] (ORTIz et a!. 1999; EvANs et aL 2007). Okpl does not display synthetic lethality with any of the SAC components in yeast. Our data show that hcp-1(RNAz), but not hcp-2(RNAz), enhances the lethality of the SAC mutants, suggesting for the first time that HCP- 1 and HCP-2 do not have completely redundant functions (TARAIL0 et al. 2007b). HCP-l and HCP-2 share only 54% sequence similarity and HCP-2 lacks the tandem repeats observed in HCP-1 and CENP-F (MooRE et a!. 1999). In CENP-F, these tandem repeats are required for strong CENP-F-kinetochore interaction (ZHu 1999). STEAR and RoTH (2004) proposed that the checkpoint pathway, unlike the chromosome segregation machinery, may not require the functions of both HCP-l/CeCENP-F and HCP-2. The data presented here demonstrate a role for HCP-1, but not HCP-2 in checkpoint function. Like in human cells, hcp-1/CENP-F appears to function upstream of the MDF-l, MDF 2 and SAN-i. In the mdf-2(vcl5) mutant strain depletion of HCP-l increases mis-segregation tenfold; suggesting that, HCP- 1 may also have a role in chromosome segregation that is independent of HCP-2. All of the suppressors bypassed the requirement for both HCP-2 and MDF- 1, but not for HCP- 1. The data in this thesis are consistent with a role for HCP- 1 in both chromosome segregation and the checkpoint. This thesis confirms the functionally conserved genetic interactions. Similar to protein interaction maps (MArrHEws et a!. 2001) the genetic interaction maps from one species are useful in predicting interactions in another species and providing insight into the function of uncharacterized proteins. Presented here is the first evidence linking the nine interologs to genome stability in C. elegans (Table 4.3). In addition, the data presented in this thesis describe the usefulness of an approach based on conservation of interacting pathway. ZH0NG and  109 TABLE 4.3 C. elegans genes that enhance the mdf-1(gk2) lethality when inactivated by RNAi Yeast  C. elegans  Gene  Gene  SWC5  F39H1 1.1  Function in S. cerevisiae  SWRI complex, exchanges histone variant H2AZ for  Function in C. elegans  mdf-i(gk2) enhancer  chromatin-bound histone H2A HCM1  Jkh-i0  Transcription factor, drives S-phase specific expression  mdf4(gk2) enhancer  of genes involved in chromosome segregation and  mdf-2(tm2190) enhancer  spindle dynamics CSM3  F23C8.9  Required for accurate chromosome segregation during  mdfI(gk2) enhancer  meiosis PAC2  GIM4  CINI  DCC]  K07H8. 1  pfd-2  Fl 6D3. 4  K09H9.2  Microtubule effector, binds aipha-tubulin, null mutant  mdf-1(gk2) enhancer  exhibits cold-sensitive microtubules and sensitivity to  mdj2(tm2 190) enhancer  benomyl  san-i (ok1580)  Subunit of the heterohexameric cochaperone prefoldin  mdf- I (gk2) enhancer  complex which binds specifically to cytosolic  mdJ2(tm2 190) enhancer  chaperonin and transfers target proteins to it  san-1(ok1580)  Tubulin folding factor D involved in beta-tubulin  mdf-i (gk2) enhancer  folding; isolated as mutant with increased chromosome  mdf-2(tm2190) enhancer  loss and sensitivity to benomyl  san-i (ok1580)  Subunit of a complex with  Ctf8p and Ctfl 8p that  shares some components with Replication Factor C,  mdJI(gk2) enhancer mdJL2(tm2 190) enhancer  required for sister chromatid cohesion and telomere length maintenance RSM24  Y39B6A.39  Mitochondrial ribosomal protein of the small subunit  mdJl(gk2) enhancer mdf-2(tm2 190) enhancer  PAP2  F3 I C3 .2  Catalytic subunit of TRAMP (Trf4/Pap2p-Mtr4p-  mdf-i (gk2) enhancer  Air lp/ p), a nuclear poly (A) polymerase complex, 2 involved in mitotic chromosome condensation, mitotic sister chromosome cohesion  STERNBERG  (2006) computationally integrated interactome data, gene expression data,  phenotype data, and functional annotation data from S. cerevisiae, D. melangoster and C.  elegans to predict genome-wide genetic interactions in C. elegans. Using the version 140 of this tool (http://tenaya.caltech.edu: 8000/predict!), of the total of 24 genetic interactions identified in  110 this study, seven were correctly predicted, which represents 29% success rate (Table 4.4). Although, programs like this would be ideal to ease the cost and labour associated with generation of genetic maps by computationally prioritizing candidates, more model organism data and better ortholog assignments are needed before this goal can be reached (ZH0NG and STERNBERG,  2006).  The conservation of pathway function in yeast and C. elegans may have an application in therapeutic approaches and is proposed to extend to human identification of genetic interologs (TARAIL0  et al. 2007b). For instance, mutations in homologs of mdf-]/MADJ, mdf-2/MAD2 and  san-]/MAD3 have been reported in a number of different tumours (reviewed in KoPs et a?. 2005). Identifying nonessential genes in humans that, like hcp-] result in synthetic lethality specifically with cancer cells exhibiting chromosome instability, but have no effect on non cancer cells may facilitate selective destruction of tumors. In this way, new potential targets for anti-tumour therapeutics can be identified. Furthermore, identification of bub-3 and hcp-] as novel interactions in C. elegans, not seen in yeast, underscores importance of pursuing such a study in this multicellular organism, which has additional gene functions.  111 TABLE 4.4 Program Predicted Interactions vs. Observed Interactions Predicted Interactions  Observed Interactions  mdf-i -F39H11.1  mdf-i +F39H11.1  mdf-l -Jkh-IO  mdf-l +Jkh-IO  mdf-2 +Jkh-1O  mdf-2 +fkh-iO  mdJ4 F23C8.9  mdf-1  +  F23C8.9  mdJi K07H8.1  mdJi  +  K07H8.1  mdf-2 + K07H8.1  mdf-2  +  K07H8.1  san-i  san-i  +  K07H8.1  -  -  K07H8.1  +  mdf-i -pfd-2  mdf-I +pfd-2  mdf-2 + pfd-2  mdf-2  mdf-2  mdf-2 + pfd-2  + pfd-2  + pfd-2  mdf-I F16D3.4  mdf I  +  F16D3.4  mdf-2  F16D3.4  mdf-2  +  F16D3.4  san-I F16D3.4  san-I  +  F16D3.4  mdf-i K09H9.2  mdf-I  +  K09H9.2  mdJ2 K09H9.2  mdJ2  +  K09H9.2  -  + -  -  -  mdf-i Y39B6A.39  mdf-I  +  Y39B6A.39  mdf-2 Y39B6A.39  mdf-2  +  Y39B6A.39  -  -  mdf-I-F31C3.2 mdf-I  mdJ4+F31C3.2  bub-3  mdf-I  +  bub-3  mdf-2 + bub-3  mdf-2  +  bub-3  san-I  bub-3  san-I  +  bub-3  hcp-l  mdfil  +  hcp-I  hcp-i  mdf-2  +  hcp-i  hcp-I  san-i  +  hcp-1  mdf-I mdf-2 san-i  —  —  —  -  —  The genetic interactions predicted by a program (ZHONG and STERNBERG, 2006) left and interactions observed in this thesis right. 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BuRKE, 1995 Checkpoint genes required to delay cell division in response to  nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 15: 6838-6844.  117 WATERs,  J. C., R. H. CHEN, A. W. MURRAY and E. D. SALMoN, 1998 Localization of Mad2 to  kinetochores depends on microtubule attachment, not tension. J. Cell Biol. 141: 1181 1191. WELLs,  W. A., and A. W. MURRAY, 1996 Aberrantly segregating centromeres activate the  spindle assembly checkpoint in budding yeast. J. Cell Biol. 133: 75-84. YANG,  Z., J. Guo,  Q. CHEN, C. DING, J. Du et al., 2005  Silencing mitosin induces misaligned  chromosomes, premature chromosome decondensation before anaphase onset, and mitotic cell death. Mol. Cell. Biol. 25: 4062-4074. ZH0NG,  W., and P. W. STERNBERG, 2006 Genome-Wide Prediction of C. elegans Genetic  Interactions. Science 311: 1481-1484. ZHU,  X., K. H. CHANG, D. HE, M. A. MANCINI, W. R. BRINKLEY et al., 1995 The C terminus of mitosin is essential for its nuclear localization, centromere/kinetochore targeting, and dimerization. J. Biol. Chem. 270: 19545-19550.  ZHU,  X., 1999 Structural requirements and dynamics of mitosin-kinetochore interaction in M phase. Mol. Cell Biol. 19: 10 16-1024.  118  Chapter 5: Conclusions 5.1  DISCUSSION  The goal of this thesis has been to obtain a better understanding of the molecular mechanisms involved in chromosome stability. More than 100 years ago, Theodor Boveri described the detrimental effect of unequal chromosome segregation on see urchin eggs and their progeny (B0vERI 1902). Boveri further postulated (in the footsteps of VON HANsEMANN 1890) that mis-segregation of chromosomes may be a cause of tumour development and birth defects (B0vERI  1914). Now it is commonly accepted that during meiosis, gain or loss of even a single  chromosome results in embryonic lethality or offspring with severe birth defects such as Down, Patau, Edwards or Klinefelter syndromes (reviewed in HAssOLD and HuNT 2001). Furthermore, abnormal chromosome number, or aneuploidy, is the most common feature of many types of cancers, suggesting that aberrant segregation of chromosomes during mitosis may be a cause or a driving force of tumorigenesis. Therefore, understanding the mechanisms and, thus, identification of genes whose mutation leads to chromosome instability (CIN), is fundamental to understanding and preventing human disease. In eukaryotes, the spindle assembly checkpoint is the major cell cycle control mechanism that ensures fidelity of chromosome segregation in mitosis and meiosis. Defects in the SAC components generate aneuploidy. Therefore, gaining a better sense of the SAC cascade is essential for discovering the causes of CIN. In C. elegans, orthologs for five widely conserved core components of the checkpoint, mdf-1/MAD1, mdf-2/MAD2, san-i/MAD3, bub-i/ BUBJ and bub-3/BUB3 have been identified (KITAGAwA and ROSE 1999; NYsTuL et al. 2003; OEGEMA et qi. 2001; TARAILO et a!. 2007a; STEIN et al. 2007). In addition to RNAi, recent availability of knockout alleles in these checkpoint components allowed determination of the phenotypic consequences in the absence of SAC gene products (KITAGAwA and RosE 1999; STEIN et a?.  119 2007; TARAIL0 et a!. 2007b). While mdf-2/MAD2, san-1/MAD3 and bub-3/BUB3 become essential only in the presence of chemical or mutational disruptions of the mitotic spindle (KITAGAwA  and ROSE 1999; NY5TuL et al. 2003; ENcALADA et al. 2005; TARAIL0 et al. 2007b),  bub-1/BUB1 and mdJ4/MADJ are essential for embryonic viability, long-term survival and fertility under normal conditions in C. elegans (KITAGAwA and RosE 1999; DEsAI et a!. 2003). Furthermore, depletion of BUB-1 results in embryonic lethality. This may be a consequence of its essential role in kinetochore function, and independent of the checkpoint (DESAI et a!. 2003). The absence of MDF-1 leads to lethality in a homozygous strain after a few generations due to an increase in segregational errors and is proposed to play an essential and primary role in the checkpoint (KITAGAwA and ROSE 1999). For this reason I have focused my research on identif’ing genetic interactions with mdf-1. The SAC pathway prevents aneuploidy by delaying anaphase onset until all chromosomes are properly attached to microtubules at kinetochores. In the absence of MDF-1 viability can be restored by mutations in downstream effectors that result in mitotic delay (FuRuTA  et al. 2000; KITAGAwA et a!. 2002; TARAIL0 et at. 2007a). The majority of the mdf  1 (gk2) suppressors delay mitotic divisions in both the germline and early embryonic cells (TARAILO  et at. 2007a), suggesting that anaphase onset delays allow more time for proper  kinetochore attachment, thus preventing defects in chromosome segregation. This is further supported by finding that these suppressors rescued the enhanced lethality of depleted BUB-3 mdJi (gk2) animals, which is also a checkpoint component. The suppressors are proposed to function downstream of the checkpoint as known SAC effectors, emb-30/APC4 and fzy 1/CDC2O, the newly identified such-i/APC5-like gene and three new putative SAC effectors, such-2, such-3 and such-4 (Figure 5.1). There are two copies of the APC5 gene in C. elegans genome, gfl-3 and such-i (TARAIL0 et a!. 2007a). Inactivation of one copy of APC5 results in a mild mitotic phenotype, weak  120 embryonic lethality along with germline maintenance problems (DAVIS at al. 2002; TARAIL0 et  a!. 2007a) leading to proposal that APC5 does not function as a part of meiotic APC/C (DAVIS at al. 2002). However, simultaneous inactivation of both gfl-3 and such-i results in meiotic arrest, suggesting that both copies of APC5 function redundantly as part of meiotic APC/C. Similarly, APCJO gene has two copies in C. elegans genome, apc-iO/F15H1O.3 and Y48G1C.12 (TARAIL0 et al. 2007). As a result of my analysis, I discovered suppressors that function by an unknown mechanism to rescue mdf-i(gk2) lethality (TARAIL0 et a!. 2007a). In these cases neither anaphase onset delays nor securin accumulation are observed (TARAIL0 et a!. 2007a). These suppressors encode unknown functions that compensate for the MDF- 1. such-6 is the only suppressor of this type that rescues the lethality of HCP- 1 depleted mdfi (gk2) animals. Similar to mdf-2 mutants, such-6 mutants have developmental delays (STEIN et a!. 2007). The phenotype is consistent with a role for such-6 in core checkpoint function (Figure 5.1). A test of this proposal would be exposure to nocodazole. In higher eukaryotes, checkpoint signaling is more complex, since a number of additional proteins that lack clear yeast orthologues have recently been found to contribute to checkpoint activity. These include Rod (rough-deal), ZwlO (zeste white 10), CENP-E, CENP-F and recently identified TAO1 kinase (ABRIEu et al. 2000; ScAER0u et a!. 2001; ENcALADA et a!. 2005; DRAvIAM et a!. 2007). C. elegans putative orthologue of TAO 1, kin-i8, is located in the region on chromosome III, where such-6 was positioned. However, sequencing of this suppressor strain revealed no nucleotide changes in the  kin-i 8 candidate. The Class II suppressors need not be components of the SAC pathway. For example,  such-7 is the only suppressor that eliminates DNA damage-induced apoptosis. Even though, apoptosis cannot rescue mdfi (gk2) lethality in C. elegans, it is possible that other aspects of the DNA damage response pathway account for the rescue. Thus, suppressors have a potential to  121  Y39B6A.39 OTHER fkh-1O  pfd-2  F23C8.9  F16D3.4 Tubulin Cohesion  K07H8.1  K09H9.2  such-6  OTHER APCJC  such-7 such-5  fi  emb-30 suc#1 such-2 to 4  DDR  Figure 5.1: Summary of the synthetic suppressed/enhanced genetic interactions with mdf-1. Blue  arrows represent synthetic suppressed interactions and red arrows represent synthetic enhanced  interactions. The coloured ovals group the genes with similar functions together. The function of interologs was determined based on their function in yeast (Table 4.3).  identifr not only new genes that are required for proper SAC function, but also new mechanisms of survival and fertility in the absence of MDF- 1. Many of the human tumours with a CJN phenotype are associated with weakened mitotic checkpoints (reviewed in KoPs et aL 2005). Furthermore, in the last few years, a number of genetic alterations in mitotic regulators have been reported in cancer cell lines (Table 5.1).  122 When separated from mdJ] (gk2) background suppressor mutants are viable, but most have defects in chromosome segregation. Thus, in addition to increasing our understanding of the regulation of chromosome segregation this collection of mutants may identify new genes that may be mutated in cancer cells. For example, such-2, -3 and -4 may identify previously undescribed SAC effectors, and such-6 is likely to be a new checkpoint component whereas such-S and such-7 may function outside the SAC pathway to maintain chromosome stability. All of the suppressor though viable display high levels of chromosomal instability in the mdf-1 (gk2) background (TARAIL0 et at. 2007a). Like mdf-i and bub-i in C .elegans, MAD and BUB genes are essential for growth in murine cells (D0BLEs et at. 2000; KALITsIs et al. 2000). In humans, loss of function in the SAC genes can result in tumours (Table 5.1). The tumour cell lines are viable but mis-segregate chromosomes resulting in CIN. Additional mutations may be needed to maintain the cell viability, analogous to the suppressor mutations described here. Thus, identifying genes required to restore viability in a model organism in the absence of an essential mitotic checkpoint contributes to our understanding of the development and growth of tumours. The such-4(h2168) suppressor was isolated in the dog-i (gki 0) mutator strain (TARAIL0 et at. 2007a). In humans, the dog-i homolog BRIP1/FANCJ (CHEuNG et a!. 2002) was found to be mutated in some individuals with early-onset breast cancer (CANToR et at. 2001) and a subset of patients with Fanconi anemia (LEvITus et a!. 2005; LEvRAN et at. 2005). In C. etegans, dog-i is required to maintain stability of guanine-rich DNA and when mutated or disrupted by RNAi results in a mutator phenotype with a slightly reduced brood size that accumulates mutations and cannot be maintained after 50 generations (CHEuNG et at. 2002). After the mdf-i(gk2) such 4(h21 68); dog-] (gk2) suppressor was isolated and outcrosses to remove the suppressor from dog-i (gk2) mutator background, one strain of mdf-i(gk2) such-4(h2168); dog-i (gk2) homozygotes was kept and maintained for more than three years (see APPENDIx B). Thus,  123 TABLE 5.1 SAC pathway genes mutated in human tumours Gene  Cancer-associated mutation  Altered expression in primary tumors  References  CENPF  Genetic amplification in esophageal squamous cell carcinoma cell lines  Over-expression in all cases with DNA amplification and also associated with Wilms tumors,  PIMKHAOKHAM et at. 2000; VAN DEN BOOM et at. 2003; GRuTzMANN et al. 2004; ZIRN  pancreatic ductal carcinomas and  et al.  2006  gliomas BUB I  Mutated in colon, lung and pancreatic cancer cells. Promoter hypermethylation in colon carcinoma  Reduced expression in AML Over-expressed in gastric and  CAI-IILL  et a!. 1998; IMAI et 1999 GEMMA et aL 2000;  a!.  breast cancers and in nonendometrioid endometrial  SHIcHIRI et at. 2002; LnI et 2002; HEMPEN et aL 2003;  al.  carcinomas  MORENO-BUENO et al. 2003;  GRABsCH et at. 2003; YuAN et  MADI  MAD2  Mutations in cancer cells from lymphoid, pancreas, prostate, breast and lung tissues Rare mutations have been  found in bladder and breast cancer cells BUBRI (Mad3 and Bub I hybrid)  BUB3  Mutated in colon and lymphoid cancer cells. Point mutations in mosaic variegated aneuploidy and premature chromatid separation syndrome. Promoter hypermethylation in colon carcinoma. ND  CDC2O  ND  PTTG1 (securin)  ND  RAD2I/SCC1 (Cohesin) APC3/CDC27 APC4 APC6/CDC16 APC8/CDC23  Amplified in hormonerefractory prostate tumors Mutated in colon cancer cells Mutated in colon cancer cells Mutated in colon cancer cells Mutated in colon cancer cells  Reduced expression associated with carcinogenesis in human gastric cancer and poorly differentiated tumors  CIN. Over-expressed in several tumor types, where it correlates with high E2F activity and poor patient prognosis Over-expressed in gastric and breast cancers.  Over-expressed in high-grade primary breast cancer and gastric carcinomas CIN. Over-expressed in head and neck, pancreatic, breast, gastric and ovarian cancer and in early stage lung adenocarcinoma CIN. Over-expressed in a wide range of human tumors. A marker of metastatic tumors  CIN. Over-expressed in prostate cancer ND ND ND ND  at. 2006 NOMOTO et aL 1999; HAN et at. 2000; TsuKAsAKI et aL 2001; NISHIGAKI et at. 2005 PERCY et aL 2000; HERNAND0 et aL 2001; HERNANDO et aL 2004; YUAN et at. 2006 CAHILL et aL 1998; CHAN et at. 1999; OHSHIMA et a!. 2000; SHICHIRI et at. 2002; GRABscH et at. 2003; MATSUURA et at. 2006; YUAN et at. 2006;  GRAB SCH et at. 2003; YUAN et at. 2006 Li et at. 2003; SINGHAL et at. 2003; KIM et at. 2005; OUELLET et at. 2006; YUAN at at. 2006; RAMASWAMY at at. 2003; GRUTzMANN et at. 2004; GENKAI et aL 2006; FUJII et at. 2006; PORKKA et at. 2004 WANG WANG WANG WANG  et aL et at. et aL et at.  2003 2003 2003 2003  The table was modified from KoPs et al. (2005) and PEREz DE CASTRO et al. (2007).  124 despite the C1N and mutator phenotype the mdf-](gk2) such-4(h2]68), dog-i (gk2) homozygotes survived for more than 270 generations and display better viability that the original mdf-i(gk2) such-4(h2168) suppressor strain (see APPENDIX B). In an attempt to identify upstream components of the checkpoint, enhancers of mdf 1 (gk2) lethality were analyzed. In yeast, many of the genes that are involved in chromosome segregation encode components of the mitotic spindle or kinetochore and are non-essential in the presence of a functional checkpoint (TONG et al. 2004; MEAsDAY and HIETER 2004; reviewed in BAETz  et al. 2006). In yeast, more than 60 kinetochore proteins have been identified (reviewed in  McAINsH  et al. 2003), and the number of proteins functioning at the mammalian kinetochore is  predicted to be more than 100 (FuKAGAwA et al. 2004). In contrast, in C. elegans less than 30 kinetochore proteins have been identified (reviewed in OEGEMA and HYMAN 2006). All of these genes are essential, except for mdf-2, san-i, bub-3, hcp-], hcp-2 and kbp-5. Putative orthologs of the non-essential yeast kinetochore components that display synthetic lethal phenotype with MAD] could not be identified by sequence comparisons reinforcing the importance of functional tests. The finding that hcp-1 enhanced the lethality of all the tested SAC components, was the first demonstration that mitotic kinetochore components in C. elegans interact with the checkpoint. Thus, large-scale screens for synthetic enhancers of the SAC components have a great potential to identify additional kinetochore components (Figure 5.1). Identification of non-essential gene functions that when absent result in synthetic enhanced/lethal phenotype with mutant SAC components has the potential to identify new, more efficient anti-tumour targets. While the defects in genes required for accurate chromosome distribution contribute to tumorigenesis, the common chemotherapeutic strategy is chronic activation of the checkpoint (reviewed in KoPs et al. 2005). 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IMOTO et a!., 2000 Nonrandom  chromosomal imbalances in esophageal squamous cell carcinoma cell lines: possible involvement of the ATF3 and CENPF genes in the 1q32 amplicon. Jpn J Cancer Res. 91: 1126-1133. P0RKKA,  K. P., T. L. TAMMELA, R. L. VEssELLA and T. VIsAK0RPI, 2004 RAD21 and KIAAO 196  at 8q24 are amplified and overexpressed in prostate cancer. Genes Chromosomes Cancer 39: 1-10. RAMASWAMY,  S., K. N. Ross, E. S. LANDER and T. R. G0LuB, 2003 A molecular signature of  metastasis in primary solid tumors. Nat. Genet. 33: 49-54. SCARE0U,  F., D. A. STARR, F. PIANO, 0. PAP0uLAs, R. E. KAREsS eta!., 2001 The ZW1O and  Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore. J. Cell Sci. 114: 3103-3114. SHIcHIRI,  M., K. YOsHINAGA, H. HISAT0MI, K. SUGIHARa and Y. HIRATA, 2002 Genetic and  epigenetic inactivation of mitotic checkpoint genes hBUB1 and hBUBR1 and their relationship to survival. Cancer Res. 62: 13-17.  132 Sn’.TGHAL,  S., K. M. AMIN, R. KRuKLITIs, P. DEL0NG, M. E. FRIscIA et a!., 2003 Alterations in  cell cycle genes in early stage lung adenocarcinoma identified by expression profiling. Cancer Biol. Ther. 2: 29 1-298 STEIN,  K. K., E. S. DAvIS, T. HAYs and A. GoLDEN, 2007 Components of the Spindle Assembly Checkpoint Regulate the Anaphase-Promoting Complex During Meiosis in Caenorhabditis elegans. Genetics 175: 107-123.  TARAIL0,  M., R. KurAGAwA and A. M. RosE, 2007a Suppressors of spindle checkpoint defect  (such) mutants identify new mdf-]/MAD1 interactors in Caenorhabditis elegans. Genetics 175: 1665 TARAIL0,  —  1679.  M., S. TARAILO and A. M. RoSE, 2007b Synthetic Lethal Interactions Identify  Phenotypic “Interologs” of the Spindle Assembly Checkpoint Components. Genetics, th, September 27 2007 (22 pages).  ToNG,  A. H.., G. LESAGE, G. D. BADER, H. DING, H. Xu et a?., 2004 Global mapping of the yeast genetic interaction network. Science 303: 808-8 13.  TsuKASAKI,  K., C. W. MILLER, B. GREENSPuN, S. ESHAGHIAN, H. KAWABATA eta?., 2001  Mutations in the mitotic check point gene, MAD1L1, in human cancers. Oncogene 20: 3301-3305. VON HAN5EMANN,  D., 1890 Uber asymmetrische Zeliheilteilungen in epithelkrebsen und deren  biologische bedeutung. Virschows. Arch. Pathol. Anat. 119: 299-326. VAN DEN BooM,  J., M. W0LmR, R. KuIcK, D. E. MISEK, A. S. YOuKILIs et a?., 2003  Characterization of Gene Expression Profiles Associated with Glioma Progression Using Oligonucleotide-Based Microarray Analysis and Real-Time Reverse Transcription Polymerase Chain Reaction. Am. J. Pathol. 163:1033-1043.  133 WANG,  Q.,  C. MOYRET-LALLE, F. CouzoN, C. SuRBIGuET-CLIPPE, J. C. SAuRIN et al., 2003  Alterations of anaphase-promoting complex genes in human colon cancer cells. Oncogene 22: 1486-1490. YuAN, B., Y. Xu, J. H. Woo, Y. WANG, Y. K. BAE et al., 2006 Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability. Clin. Cancer Res. 12: 405-410. ZIRN, B., 0. HAwrMANN, B. SAMANs, M. KRAUSE, S. WITrMANN et at., 2006 Expression profiling of Wilms tumors reveals new candidate genes for different clinica l parameters.  mt. J. Cancer 118:  1954-1962  134  APPENDIX A.  Chromosome V GIC-tract analysis in such-4(h2168) suppressor strain The such-4(h2i68) suppressor, isolated using the dog-i (gki 0) mutato r strain (CHEuNG  et a!. 2002), was positioned to gene cluster on chromosome V. There are 57 runs of G/C of 1 8bp or more located on chromosome V. To test whether any of the polyguanine tracts are deleted in such-4(h2168) suppressor strain, primers spanning a G/C-tract were design ed and the region was amplified using the standard PCR procedure. In order to distinguish betwee n recent and inherited G-tract deletions, three bulked lysates were formed. First contained 100 mdJi (gk2) such 4(h2168) worms, second contained 100 mdf-1(gk2) such-4(h2168); dog-i (gk2) control worms  and third contained 100 wild-type control worms. In total 44 G/C-tracts of l8bp and longer located on chromosome V were analyzed (Table A.1). The mdJ4(gk2) such-4 (h2168); dog i(gk2) strain that was kept homozygous for more than 40 generations, at the time of the analysis, had multiple G/C-tract deletions (Table A. 1), consistent with the absence of DOG-i (CHEuNG et al. 2002). Furthermore, the strain was homozygous for 1 8Obp deletion in a G-tract located upstream of unc-34 gene in the Y5OD4C cosmid. However, neither the wild-ty pe control nor the mdf-1(gk2) such-4(h2168) strain had any of the G/C-tract deletions detected in the mdJl(gk2) such-4(h2i68); dog-i(gk2) (Table A.1). First, these data suggest that the inherited l8Obp deletion in the Y5OD4C G-tract was generated after the mdf-i(gk2) such-4(h2168 ) suppressor strain was out-crossed from the dog-i (gk2) background (see Chapter 2 MATE RIALS and METHODS). Second, it is possible that lesion in the mdf-i(gk2) such-4(h2168) suppressor strain is located in a G-rich sequence other than polyguanine runs longer than 1 8bp. It is also possible that DOG-i may have additional uncharacterized targets. Furthermore, the analysis of the mdf 1(gk2) such-4(h2]68); dog-i (gk2) strain revealed that not all polyguanine tracks in genomic DNA larger than 1 8bp are vulnerable to deletion events (Table A. 1). This is consistent with the model and findings reported by Ci-iEuNG et a!. (2002).  135  Table A.1 Analysis of G/C-tracts, located on chromosome V, in such-4(h2168) suppressor strains Tract Location F3 1 F4 T22H9 Y5OD4C Y5 OD4C Y75B7AL F52F10 R12A1 TO6A1 F59A7 C44C3 F59D6 C5OH1 1 C5OH1 1 Y73C8B K08D9 K08D9 Cl 7E7 Y45G5AM T28F12 T28F 12 T28F12 F54D1 1 C37H5 Y49G5B B0238 ZC404 KO9G1 D2023 R13H4 F58D12 Y5OE8A C30G7 C55A1 C06C6 F44G3 F14F8 Y2OC6A F59A1 C47A10 Y59A8B Y51A2D Y69H2 Y39B6A F3 8A6  Gene  N2  mdf-i(gk2) such-4(h2168)  mdf-i(gk2) such-4(h2168); dog-i (gk2)  sru-24  No No No  No deletion bands No deletion bands No deletion bands  No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No  No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands  Deletions observed Deletions observed, multiple Homozygousfor i8Obp deletion ofthis G-tract Deletions observed Deletions observed No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands No deletion bands Deletions observed, multiple Deletions observed, multiple No deletion bands No deletion bands No deletion bands No deletion bands Deletions observed Deletions observed Deletions observed, multiple No deletion bands No deletion bands No deletion bands Deletions observed No deletion bands No deletion bands No deletion bands No deletion bands Deletions observed, multiple No deletion bands Deletions observed No deletion bands Deletions observed No deletion bands No deletion bands No deletion bands Deletions observed No deletion bands No deletion bands No deletion bands No deletion bands Deletions observed Deletions observed, multiple  F52F10.2  F59D6.7  apx-1 Y45G5AM.2 unc-62 unc-62  C37H5.3 B0238.7  C55A1.1  srw-42  sre-21 hmit-1.1  pha-4  136 B.  Phenotypic characterization of the such-4(h2168) suppressor strains After the mdfi(gk2) such-4(h2168), dog-i (gk2) suppressor was isolated and  outcrosses to remove the suppressor from dog-i (gk2) mutator background, one mdf-i( gk2) such 4(h2168); dog-i (gk2) strain was maintained. dog-i was shown to accumulate mutati ons resulting in difficulty to maintain the strain after 50 generations (CHEuNG et a!. 2002). Howev er, mdf i(gk2) such-4(h2i68); dog-i (gk2) homozygotes survived for more than 270 generations, which prompted phenotypic analysis of such-4(h2168) suppressor strains (Table A.2). This analysis revealed that KR4250 strain has better viability than KR4233 strain, while the KR4460 strain, mdf-](gk2) such-4(h2168); dog-] (gk2) homozygotes maintained for more than 270 genera tions, have generally the best viability, when compared to all other strains (Figure A. 1). These data suggest the possibility that there are additional DOG-i targets that when mutated rescue the lethality of mdf-i (gk2) worms. Thus, it would be very useful to analyze the spectrum of deletio ns and potentially other lesions in the KR4460 strain, using a method such as array comparative genomic hybridization (MAYDAN et al. 2007).  Table A.2 The such-4(h2168) strains Strain  Genotype  KR4233  unc-46(e177) mdf-1(gk2) such-4(h2168)  KR425 0  unc-46(el 77) mdf-1 (gk2) such-4(h2 168); dog-i (gkl 0) (F68)  KR4460  unc-46(e177) mdf-1(gk2) such-4(h2168); dog-I (gkl 0) (F270)  The KR4250 and KR4460 strains are also homozygous for 1 8Obp deletion in the Y5OD4C G-tract.  137  100 90 0’  >  50 >  30 20 10 0 Hatch  Adult  I unc-46 mdf-i such-4 (F7) 0 unc-46 mdf-l such-4; dog-i (F68)  0 unc-46 mdf-1 such-4; dog-I (F270) Figure A.1: Viability of three different such-4(h2168) suppressor strains. The unc-46(e177) mdf  i(gk2) such-4(h2168) strain was analyzed seven generations after it was out-crossed from dog-i (gki 0) background. One unc-46(ei 77) mdf-i (gk2) such-4(h2i 68), dog-i (gki 0) strain was kept on plates and analyzed after 68 generations. After analysis one plate from this strain was frozen under KR4250 number, another plate was kept further and analyzed after 270 generations. After the analysis these worms were frozen as well under KR4460 number.  138 C.  BIBLIOGRAPHY  CHEuNG,  I., M. ScHERTzER, A. M. ROSE and P. M. LANsD0RP, 2002 Disruption of dog-i in  Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA. Nat. Genet. 31: 405-409. MAYDAN, J.  S., S. FLIBOTTE, M. L. EDGLEY, J. LAu, R. R. SELzER et al. 2007 Efficient high-  resolution deletion discovery in Caenorhabditis elegans by array comparative genomic hybridization. Genome Res. 17: 337-47  Yahoo! 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